Methods for using extracellular adenosine inhibitors and adenosine receptor inhibitors to enhance immune response and inflammation

ABSTRACT

A method is provided herein to increase an immune response to an antigen. The method includes administering an agent that inhibits extracellular adenosine or inhibits adenosine receptors. Also disclosed are methods to increase the efficacy of a vaccine and to increase an immune response to a tumor antigen or immune cell-mediated tumor destruction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/310,264 filed Dec. 2, 2011, which is a divisional of U.S. applicationSer. No. 10/498,416 filed Jun. 10, 2004, U.S. Pat. No. 8,080,554, whichis the U.S. National Stage of International Application No.PCT/US02/36829 filed Nov. 14, 2002, which claims priority to U.S.Provisional Application Nos. 60/340,772 filed Dec. 12, 2001 and60/342,585 filed Dec. 19, 2001, all hereby incorporated by reference intheir entirety.

FIELD

This application relates to the use of inhibitors of extracellularadenosine and/or inhibitors of adenosine receptors, such as adenosinereceptor antagonists and agents that decrease formation or degradeextracellular adenosine, to enhance an immune response and inflammation,and in some examples modulate NF-kB activity.

BACKGROUND

The inflammatory response helps eliminate harmful agents from the body,but inflammation is also a non-specific response that can harm healthytissue. There is a wide range of pathogenic insults that can initiate aninflammatory response including infection, allergens, autoimmunestimuli, immune response to transplanted tissue, noxious chemicals, andtoxins, ischemia/reperfusion, hypoxia, mechanical and thermal trauma, aswell as growth of tumors. Inflammation is normally a localized actionthat results in expulsion or dilution of a pathogenic agent, resultingin isolation of the damaging agent and injured tissue. The cellsinvolved in inflammation include leukocytes (i.e. the immune systemcells—neutrophils, eosinophils, lymphocytes, monocytes, basophils,macrophages, B cells, dendritic cells, granulocytes and mast cells), thevascular endothelium, vascular smooth muscle cells, fibroblasts, andmyocytes.

Adenosine modulates diverse physiological functions including inductionof sedation, vasodilatation, suppression of cardiac rate andcontractility, inhibition of platelet aggregability, stimulation ofgluconeogenesis and inhibition of lipolysis (see, Stiles, TrendsPharmacol. Sci. 7:486, 1986; Williams, Ann. Rev. Pharmacol. Toxicol.27:315, 1987; Ramkumar et al., Prog. Drug. Res. 32:195, 1988). Inaddition, adenosine and some adenosine analogs that non-selectivelyactivate adenosine receptor subtypes decrease neutrophil production ofinflammatory oxidative products (Cronstein et al., Ann. N.Y. Acad. Sci.451:291, 1985; Roberts et al., Biochem. J., 227:669, 1985; Schrier etal., J. Immunol. 137:3284, 1986; Cronstein et al., Clinical Immunol.Immunopath. 42:76, 1987).

Based on biochemical and pharmacological criteria, four subtypes ofadenosine receptors have been differentiated: A2a, A2b, A1, and A3. A1and A3 inhibit, and A2a and A2b stimulate, adenylate cyclase,respectively (Stiles, ibid; Williams, ibid; see also U.S. Pat. No.5,441,883 for A3 receptors). Substantial progress has been madeconcerning the biochemical and pharmacological properties of theseadenosine receptors such as ligand binding characteristics,glycosylation, and regulation. In addition to its effects on adenylatecyclase, adenosine opens potassium channels, reduces flux throughcalcium channels, and inhibits or stimulates phosphoinositide turnoverthrough receptor-mediated mechanisms (Fredholm and Dunwiddie, TrendsPharmacol. Sci. 9:130, 1988; Sebastiao et al., Br. J. Pharmacol. 100:55,1990; Stiles, Clin. Res. 38:10, 1990; and Nakahata et al., J. Neurochem.57:963, 1991). The cDNAs that encode the A1, A2, and A3 adenosinereceptors have been cloned (Libert et al., Science 244:569, 1989;Maenhaet et al., Biochem. Biophys. Res. Commun. 173:1169, 1990; Libertet al., EMBO J. 10:1677, 1991; Mahan et al., Molecular Pharmacol. 40:1,1991; Reppert et al., Molec. Endo. 5:1037-1048, 1991; U.S. Pat. No.5,441,883). Molecular cloning of the adenosine receptors has revealedthat they belong to the superfamily of G-protein coupled receptors.

SUMMARY

It is disclosed herein that adenosine receptors play a non-redundantrole in down-regulation of inflammation in vivo by acting as aphysiological “STOP” (a termination mechanism) that can limit the immuneresponse and thereby protect normal tissues form excessive immune damageduring pathogenesis of different diseases. Adenosine receptors, such asA2a, A2b, and A3, are shown to down-regulate the immune response duringinflammation and protect tissues from immune damage. Inhibition ofsignaling through the adenosine receptor can be used to intensify andprolong the immune response.

Methods are provided herein to increase an immune response. In oneexample, the method increases desirable and targeted tissue damage, suchas damage of a tumor, for example cancer. Disclosed herein are methodsof inhibiting one or more processes conducive to the production ofextracellular adenosine and adenosine-triggered signaling throughadenosine receptors. For example, enhancement of an immune response,local tissue inflammation, and targeted tissue destruction isaccomplished by: inhibiting or reducing the adenosine-producing localtissue hypoxia; by degrading (or rendering inactive) accumulatedextracellular adenosine; by preventing or decreasing expression ofadenosine receptors on immune cells; and/or by inhibiting/antagonizingsignaling by adenosine ligands through adenosine receptors. The resultsdisclosed herein demonstrate that by in vivo administration of agentsthat disrupt the “hypoxia->adenosine accumulation->immunosuppressiveadenosine receptor signaling to immune cells” pathway in subjectssuffering from various diseases (e.g. cancer and sepsis) can result inin vivo treatment of tumors or improved immunization.

In one example, the method includes administering one or more inhibitorsof extracellular adenosine and/or adenosine receptor inhibitors, such asan adenosine receptor antagonist. To increase the efficacy of a vaccine,one or more adenosine receptor inhibitors and/or inhibitors ofextracellular adenosine can be administered in conjunction with thevaccine. In one example, one or more adenosine receptor inhibitors orinhibitors of extracellular adenosine are administered to increase animmune response/inflammation. In another example, a method is providedto achieve targeted tissue damage, such as for tumor destruction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a bar graph showing that pharmacologically activatedcAMP-elevating receptors or increases in cAMP are capable of blockinginflammation in vivo. The differences between treated and untreated miceare statistically significant as indicated by the asterisk (*P<0.05).

FIG. 1B is a bar graph showing that pharmacologically activatedcAMP-elevating A2a receptors are capable of blocking inflammation invivo. The differences between treated and untreated mice arestatistically significant as indicated by the asterisk (*P<0.05).

FIGS. 2A and 2B are bar graphs showing cAMP levels in lymphoid cellsfrom (A) wild-type (A2aR+/+) or (B) A2aR deficient (A2aR^(−/−)) micetreated with CGS21680 alone, or CGS21680 and ZM241385.

FIGS. 2C and 2D are bar graphs showing cAMP levels in lymphoid cells in(C) wild-type (A2aR+/+) or (D) A2aR deficient (A2aR^(−/−)) treated withFK, isoproterenol, or PGE₂. The differences between treated anduntreated mice are statistically significant as indicated by theasterisk (*P<0.05).

FIGS. 3A and 3B are dot plots showing serum levels of (A) ALT or (B)TNF-α in A2aR^(+/+) and A2aR^(−/−) mice at various time points.

FIG. 4A is a bar graph showing serum ALT levels in mice treated withdifferent combinations of inflammatory stimuli,(Con-A) and A2 receptorantagonist ZM241385. *P<0.05 versus A2aR^(+/+) mice.

FIGS. 4B and 4C are scatter plots showing serum ALT levels in miceinjected with (B) Pseudomonas Exotoxin A or (C) carbon tetrachloride.

FIG. 5 is a survival graph showing that A2a receptors protect againstdeath from septic shock. *P<0.05.

FIG. 6A-D are scatter plots showing the serum levels of the indicatedcytokines in A2aR^(−/−) mice as compared with A2aR^(+/+) wild type micesubjected to endotoxic shock. *P<0.05.

FIG. 7 is a digital image showing the results of a ribonucleaseprotection assay (RPA) demonstrating the increase of inflammation (bypro-inflammatory cytokines mRNA expression) in mice with inactivated A2areceptors, after treatment with inflammatory stimuli.

FIGS. 8A and 8B are digital images showing a ribonuclease protectionassay (RPA) demonstrating the increase of inflammation (bypro-inflammatory cytokines mRNA expression) in mice withpharmacologically inactivated A2a receptors after treatment withinflammatory stimuli.

FIG. 9 is a digital image showing the results of a nuclear extractelectrophoretic mobility shift assay of macrophages that demonstratesthat A2a receptors negatively regulate NF-kB translocation into thenucleus, and thereby its activity, in vivo.

FIGS. 10 A and 10B are digital images of Western blots showing thatadenosine receptors negatively regulate NF-kB translocation byinhibiting phosphorylation of Ik-B by IKK kinase.

FIG. 11 is a schematic showing the intracellular events following theactivation of immune cells and the mechanism of A2a adenosine receptorand cAMP-mediated inhibition of NF-kB activities.

FIGS. 12A-12C are dot plots showing that adenosine receptor antagonistsimprove immunotherapy of cancer tumors by reducing the number ofmetastatic nodules.

FIGS. 13 and 14 are graphs showing that adenosine receptor antagonistsimprove the therapy of cancers by reducing the size/volume of tumors.

FIG. 15 is a scatter plot showing IgG₁ concentration in mice injectedsubcutaneously with TNP-KLH alone (CFA) or with adenosine receptorantagonist theophylline (CFA+antagonist) and demonstrates theimprovement of antibody production by theophylline in the immunizationmixture.

FIG. 16 is a summary of the methods that can be used to enhance and/orprolong inflammatory responses by inhibiting or decreasing theendogenous anti-inflammatory processes.

SEQUENCE LISTING

The nucleic acid sequence listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand.

SEQ ID NO: 1 shows the nucleotide sequence of a CpG oligonucleotide.

DETAILED DESCRIPTION OF SEVERAL SPECIFIC EMBODIMENTS Abbreviations

Adora1 Adenosine receptor A1Adora2a Adenosine receptor A2aAdora2b Adenosine receptor A2bAdora3 Adenosine receptor A3ADA Adenosine deaminase, adenosine degrading enzymeADA-PEG Polyethylene glycol-modified ADA to extend half life in vivoADA SCID Adenosine deaminase severe combined immunodeficiencyALT Alanine aminotransferase. Liver enzymecAMP Cyclic adenosine monophosphate

CGS CGS21680 Con A: Concanavalin A FK Forskolin

H-E Haematoxylin and eosin

IL-12p40 Interleukin-12p40 IL-1β Interleukin-1β IL-6 Interleukin-6 IsoIsoproterenol

LPS Lipopolysaccharide, bacterial endotoxinPEA Pseudomonas exotoxin A

PGE₂ Prostaglandin E2

TNF-α Tumor necrosis factor α

Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein and inthe claims, the singular forms “a” or “an” or “the” include pluralreferences unless the context clearly dictates otherwise. For example,reference to “an adenosine receptor antagonist” includes a plurality ofsuch antagonists and reference to “the adenosine receptor” includesreference to one or more receptors and equivalents thereof known tothose skilled in the art, and so forth. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

Adenosine: A ribonucleotide which includes the nitrogenous base,adenine, linked to the sugar, ribose.

Adenosine receptors: At least four subtypes of adenosine receptor(Adora1, Adora2a, Adora2b, and Adora3, also called A1, A2a, A2b, and A3,respectively) have been cloned. Adenosine receptors include bothnaturally occurring peptides, as well as adenosine receptor fragmentsand variants that retain full or partial adenosine receptor biologicalactivity. Adenosine receptors are members of the G-protein coupledreceptor (GPCR) superfamily, and are thought to mediate stimulation orinhibition of adenylyl cyclase activity, and hence cyclic AMP levels.The effects on cAMP levels of the methylxanthine antagonists ofadenosine receptors (such as caffeine, and theophylline which arepresent in tea, coffee and cocoa) are known.

The A1 receptor is linked to inhibition of adenylyl cyclase activity.However, there is also evidence for coupling (via G-proteins) to ionchannels, and phospholipase C. In the nervous system, the A1 adenosinereceptor mediates inhibition of transmitter release and the reduction inneuronal activity. Blockade of this receptor in the heart leads to theaccelerated, pronounced “pounding” observed after drinking large amountsof strong coffee (due to caffeine and theophylline). In one example, A1is shown as GenBank Accession No. L22214.

A2a is almost exclusively coupled to stimulation of adenylyl cyclaseactivity. Its distribution in the CNS is very discrete, being heavilylocalized in the caudate and putamen bodies, and the nucleus accumbensand olfactory tubercle. In the periphery, the A2a receptor is present onplatelets and is anti-aggregatory. In one embodiment, A2A is shown asGenBank Accession No. AH003248, and A2b is shown as GenBank AccessionNo. NM000676. A2a and A2b receptors cause similar cellular effects, buthave different tissue distribution and requirements for the levels ofextracellular adenosine needed for their activation. It appears that A2breceptor is activated by higher levels of adenosine then A2a receptor(Linden, Ann. Rev Pharmacol. Toxicol. 41:775-87, 2001).

A3 couples to inhibition of adenylyl cyclase activity. In one example,A3 is shown as GenBank Accession No. AH003597.

Adenosine receptor inhibitor: Any agent or composition that decreasesthe activity of an adenosine receptor. For example, such an inhibitormay decrease the activity of an adenosine receptor, as compared to theactivity of the adenosine receptor in the absence of such an inhibitor.Examples include, but are not limited to, a pharmacological antagonist,a gene therapy agent, a ribozyme, an antisense oligonucleotide, oranother catalytic nucleic acid that selectively binds mRNA encoding anadenosine receptor.

Adjuvant: Any agent that enhances or increases one or moreimmune-stimulating properties of another agent (such as a chemicalcompound or antigenic epitope). An adjuvant augments, stimulates,activates, potentiates, or modulates the immune response at the cellularor humoral level.

For example, addition of an adjuvant to a vaccine improves the immuneresponse of a cell, such as a cell in a subject. An adjuvant can be usedso that less vaccine is needed to produce the immune response. Onespecific, non-limiting example of an adjuvant is Freund's adjuvant,which is a water-in-oil emulsion that contains an immunogen, anemulsifying agent and mycobacteria. The classical agents (Freund'sadjuvant, BCG, Corynebacterium parvum) contain bacterial antigens. Someadjuvants are endogenous (e.g. histamine, interferon, transfer factor,tuftsin, interleukin-1 and interleukin-12). The mode of action of anadjuvant can be non-specific, resulting in increased immuneresponsiveness to a wide variety of antigens, or antigen-specific, i.e.affecting a restricted type of immune response to a narrow group ofantigens. The therapeutic efficacy of many biological response modifiersis related to their antigen-specific immunoadjuvanticity.

Agent: Any polypeptide, compound, small molecule, organic compound,salt, polynucleotide, peptidomimetic, or other molecule of interest.

Agonist: An agent that has affinity for and stimulates physiologicactivity of a receptor normally stimulated by one or more naturallyoccurring agents, thus triggering a biochemical response. In oneexample, one molecule of agonist (A) binds reversibly to a receptormolecule (R) to form an active agonist-receptor complex (AR), whichgenerates a pharmacological response while the agonist remains bound.

Antagonist: An agent that tends to nullify the action of another, as adrug that binds to a receptor without eliciting a biological response.In one example, an antagonist is a chemical compound that is anantagonist for an adenosine receptor, such as the A2a, A2b, or A3receptor. Specific examples of adenosine receptor antagonists, include,but are not limited to: ZM241385; MRS1220; 1,7, methylxantine(caffeine); theophylline; teobromin; SCH 58261[7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine](Schering-Plough Research Institute, Milan, Italy); KW-6002[(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione](Kyowa Hakko Kogyo Co. Ltd., Shizuoka, Japan); and ADA-PEG. Particularnon-limiting examples of antagonists are described in U.S. Pat. Nos.5,565,566; 5,545,627, 5,981,524; 5,861,405; 6,066,642; 6,326,390;5,670,501; 6,117,998; 6,232,297; 5,786,360; 5,424,297; 6,313,131,5,504,090; and 6,322,771. In another example, an adenosine receptorantagonist is an antisense oligonucleotide, ribozyme, or other catalyticnucleic acid that selectively binds mRNA encoding the adenosinereceptor.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e. molecules that contain anantigen-binding site that specifically binds (immunoreacts with) anantigen. A naturally occurring antibody (e.g. IgG) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. However, the antigen-bindingfunction of an antibody can be performed by fragments of anaturally-occurring antibody. Thus, these antigen-binding fragments arealso designated by the term “antibody”.

Examples of binding fragments encompassed within the term antibodyinclude (i) an Fab fragment consisting of the VL, VH, CL and CH1domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (iv) a dAb fragment (Ward et al., Nature 341:544-6, 1989)which consists of a VH domain; (v) an isolated complimentaritydetermining region (CDR); and (vi) an F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region. Furthermore, although the two domains of the Fvfragment are coded for by separate genes, a synthetic linker can be madethat enables them to be made as a single protein chain (known as singlechain Fv (scFv); Bird et al. Science 242:423-6, 1988; and Huston et al.Proc. Natl. Acad. Sci. 85:5879-83, 1988) by recombinant methods. Suchsingle chain antibodies are also included.

In one example, antibody fragments are those which are capable ofcrosslinking their target antigen, e.g., bivalent fragments such asF(ab′)₂ fragments. Alternatively, an antibody fragment which does notitself crosslink its target antigen (e.g., a Fab fragment) can be usedin conjunction with a secondary antibody which serves to crosslink theantibody fragment, thereby crosslinking the target antigen. Antibodiescan be fragmented using conventional techniques and the fragmentsscreened for utility in the same manner as described for wholeantibodies. An antibody is further intended to include bispecific andchimeric molecules that specifically bind the target antigen.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen, such as a T cell surfacemolecule. The binding is a non-random binding reaction between anantibody molecule and an antigenic determinant of the T cell surfacemolecule. The desired binding specificity is typically determined fromthe reference point of the ability of the antibody to differentiallybind the T cell surface molecule and an unrelated antigen, and thereforedistinguish between two different antigens, particularly where the twoantigens have unique epitopes. An antibody that specifically binds to aparticular epitope is referred to as a “specific antibody.”

Antigen: A compound, composition, or agent capable of being the targetof inducing a specific immune response, such as stimulate the productionof antibodies or a T-cell response in a subject, including compositionsthat are injected or absorbed into a subject. An antigen reacts with theproducts of specific humoral or cellular immunity, including thoseinduced by heterologous immunogens. The term “antigen” includes allrelated antigenic epitopes.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse compliment), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T). Antisense molecules are molecules that arespecifically hybridizable or specifically complementary to either RNA orthe plus strand of DNA. Sense molecules are molecules that arespecifically hybridizable or specifically complementary to the minusstrand of DNA. Antigene molecules are either antisense or sensemolecules directed to a dsDNA target.

Antisense oligonucleotide: A sequence of at least about 8 nucleotides,such as about at least 10, 12, 15, 20, 30 or 50 nucleotides, wherein thesequence is from a gene sequence (such as all or a portion of a cDNA orgene sequence, or the reverse complement thereof), arranged in reverseorientation relative to the promoter sequence in a transformationvector.

In one example, the sequence is an adenosine receptor sequence (e.g.Genbank accession number L22214, AH003248, NM000676, and AH003597).Where the reverse complement of a adenosine receptor sequence is used tosuppress expression of proteins from the adenosine receptor locus, thesense strand of adenosine or adenosine receptor locus or cDNA isinserted into the antisense construct. A reduction of adenosine receptorprotein expression in a transgenic cell can be obtained by introducinginto cells an antisense oligonucleotide based on an adenosine receptorlocus, e.g. the adenosine receptor A1, A2a, A2b, or A3 locus, includingthe reverse complement of the adenosine receptor cDNA coding sequence,the adenosine receptor cDNA or gene sequence or flanking regionsthereof.

The introduced sequence need not be the full-length human adenosinereceptor cDNA or gene or reverse complement thereof, and need not beexactly homologous to the equivalent sequence found in the cell type tobe transformed. Generally, however, where the introduced sequence is ofshorter length, a higher degree of homology to the native adenosine oradenosine receptor locus sequence will be needed for effective antisensesuppression. The introduced antisense sequence in the vector can be atleast 30 nucleotides in length, and improved antisense suppression willtypically be observed as the length of the antisense sequence increases,such as when the sequence is greater than 100 nucleotides. Forsuppression of the adenosine receptor gene itself, transcription of anantisense construct results in the production of RNA molecules that arethe reverse complement of mRNA molecules transcribed from the endogenousadenosine receptor gene in the cell. For suppression of proteinexpression from the opposite strand of the adenosine receptor locus,transcription of an antisense construct results in the production of RNAmolecules that are identical to the mRNA molecules transcribed from theendogenous adenosine or adenosine receptor gene, assuming the antisenseconstruct was generated from sequence within the adenosine receptor generather than in a flanking region. Antisense molecules made to target thesequence that is the reverse complement of the adenosine receptor locuswill serve to suppress any abnormal expression of proteins or peptidesfrom the strand of the locus not encoding the adenosine receptor cDNA.

Autoimmune disorder: A disorder in which the immune system produces animmune response (e.g. a B cell or a T cell response) against anendogenous antigen, with consequent injury to tissues.

Avidity: The overall strength of interaction between two agents ormolecules, such as an antigen and an antibody. Avidity depends on boththe affinity and the valency of interactions. Therefore, the avidity ofa pentameric IgM antibody, with ten antigen binding sites, for amultivalent antigen can be much greater than the avidity of a dimericIgG molecule for the same antigen.

B cell or B lymphocyte: One of the two major types of lymphocyte. Theantigen receptor on B lymphocytes, sometimes called the B cell receptor,is a cell-surface immunoglobulin. On activation by an antigen, B cellsdifferentiate into cells producing antibody molecules of the sameantigen-specificity as this receptor.

Binding or stable binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including both functional and physicalbinding assays. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription,translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA include such methods as DNase I or chemical footprinting, gelshift and affinity cleavage assays, Northern blotting, dot blotting andlight absorption detection procedures. For example, one method that iswidely used involves observing a change in the light absorption of asolution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is anincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target disassociate from each other, ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

Biological samples: Suitable biological samples include samplescontaining genomic DNA, RNA (including mRNA), and/or protein, obtainedfrom cells of a subject. Examples include, but are not limited to,peripheral blood, urine, semen, saliva, tissue biopsy, surgicalspecimen, amniocentesis samples, derivatives and fractions of blood suchas serum, and biopsy material.

Cancer: Malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Complementarity and percentage complementarity: Molecules withcomplementary nucleic acids form a stable duplex or triplex when thestrands bind, (hybridize), to each other by forming Watson-Crick,Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when anoligonucleotide remains detectably bound to a target nucleic acidsequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strandbase pair with the bases in a second nucleic acid strand.Complementarity is conveniently described by percentage, i.e. theproportion of nucleotides that form base pairs between two strands orwithin a specific region or domain of two strands. For example, if 10nucleotides of a 15-nucleotide oligonucleotide form base pairs with atargeted region of a DNA molecule, that oligonucleotide is said to have66.67% complementarity to the region of DNA targeted.

In the present disclosure, “sufficient complementarity” means that asufficient number of base pairs exist between the oligonucleotide andthe target sequence to achieve detectable binding, and in the case ofthe binding of an antigen, disrupt expression of gene products (such asadenosine receptors). When expressed or measured by percentage of basepairs formed, the percentage complementarity that fulfills this goal canrange from as little as about 50% complementarity to full, (100%)complementary. In general, sufficient complementarity is at least about50%, for example at least 75%, 90%, 95%, 98% or even 100%complementarity.

A thorough treatment of the qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al. Methods Enzymol 100:266-285,1983, and by Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Comprises: A term that means “including.” For example, “comprising A orB” means including A or B, or both A and B, unless clearly indicatedotherwise.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one example, a cytokine is a chemokine, amolecule that affects cellular trafficking.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide. Theterm codon is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Deletion: The removal of a sequence of DNA, the regions on either sidebeing joined together.

Differentiation: The process by which cells become more specialized toperform biological functions. Differentiation is a property that istotally or partially lost by cells that have undergone malignanttransformation.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody binds a particular antigenicepitope.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence. For example, atherapeutically effective oligonucleotide can be complementary to anadenosine receptor-encoding mRNA, or an adenosine receptor-encodingdsDNA.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though waste times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11.

For the purpose of the present disclosure, “stringent conditions”encompass conditions under which hybridization will only occur if thereis less than 25% mismatch between the hybridization molecule and thetarget sequence. “Stringent conditions” can be broken down intoparticular levels of stringency for more precise definition. Thus, asused herein, “moderate stringency” conditions are those under whichmolecules with more than 25% sequence mismatch will not hybridize;conditions of “medium stringency” are those under which molecules withmore than 15% mismatch will not hybridize, and conditions of “highstringency” are those under which sequences with more than 10% mismatchwill not hybridize. Conditions of “very high stringency” are those underwhich sequences with more than 6% mismatch will not hybridize.

Hypersensitivity: Immune responses to innocuous antigens that lead tosymptomatic reactions upon re-exposure are called hypersensitivityreactions. These can cause hypersensitivity diseases if they occurrepetitively. This state of heightened reactivity to an antigen iscalled hypersensitivity. Hypersensitivity reactions are classified bymechanism: type I hypersensitivity reactions involve IgE antibodytriggering of mast cells; type II hypersensitivity reactions involve IgGantibodies against cell-surface or matrix antigens; type IIIhypersensitivity reactions involve antigen:antibody complexes; and typeiV hypersensitivity reactions are T cell-mediated.

Immune cell: Any cell involved in a host defense mechanism, such ascells that produces pro-inflammatory cytokines, and such as cells thatparticipate in tissue damage and/or disease pathogenesis. Examplesinclude, but are not limited to: T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, and eosinophils.

Immune response: A change in immunity, for example, a response of a cellof the immune system, such as a B cell or T cell, to a stimulus. In oneexample, the response is specific for a particular antigen (an“antigen-specific response”). In one example, an immune response is a Tcell response, such as a Th1, Th2, or Th3 response. In a particularexample, an increased or enhanced immune response is an increase in theability of a subject to fight off a disease, such as a viral infectionor tumor.

Immunoglobulins: A class of proteins found in plasma and other bodyfluids that exhibits antibody activity and binds with other moleculeswith a high degree of specificity; divided into five classes (IgM, IgG,IgA, IgD, and IgE) on the basis of structure and biological activity.Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g. see U.S. Pat. No.4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694;EP 125, 023; Faoulkner et al., Nature 298:286, 1982; Morrison, J.Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

A native (naturally occurring) immunoglobulin is made up of fourpolypeptide chains. There are two long chains, called the “heavy” or “H”chains which weigh between 50 and 75 kilodaltons and two short chainscalled “light” or “L” chains weighing in at 25 kilodaltons. They arelinked together by disulfide bonds to form a “Y” shaped molecule. Eachheavy chain and light chain can be divided into a variable region and aconstant region. An Fc region includes the constant regions of the heavyand the light chains, but not the variable regions.

Inhibitor of extracellular adenosine: Any agent or composition thatdecreases the activity or level of extracellular adenosine. Examplesinclude, but are not limited to, agents that degrade extracellularadenosine, render extracellular adenosine inactive, and/or decrease orprevent the accumulation or formation of extracellular adenosine.Particular examples include, but are not limited to, enzymes such asadenosine deaminase, adenosine kinase, and adenosine kinase enhancers;oxygenation; redox-potential changing agents which diminish the degreeof hypoxia-ischemia; and other catalytic agents that selectively bindand decrease or abolish the ability of endogenously formed adenosine tosignal through adenosine receptors. Other examples include cell cultureconditions that result in negative selection of cells with adenosinereceptors and enrichment of cell populations without adenosinereceptors.

Inflammation: When damage to tissue occurs, the body's response to thedamage is usually inflammation. The damage can be due to trauma, lack ofblood supply, hemorrhage, autoimmune attack, transplanted exogenoustissue, or infection. This generalized response by the body includes therelease of many components of the immune system (e.g. IL-1 and TNF),attraction of cells to the site of the damage, swelling of tissue due tothe release of fluid and other processes.

Inflammation, the response of tissue to injury, is divided into twophases, termed acute and chronic. In the acute phase, inflammation ischaracterized by increased blood flow and vascular permeability,accumulation of fluid, and accumulation of leukocytes and inflammatorymediators (e.g. cytokines). In the subacute/chronic phase, inflammationis characterized by the development of specific humoral and cellularimmune responses to the pathogen(s) present at the site of tissueinjury. During both the acute and chronic inflammatory processes, avariety of soluble factors are involved in leukocyte recruitment throughincreased expression of cellular adhesion molecules and chemoattraction.Many of these soluble mediators regulate the activation of both theresident cells (such as fibroblasts, endothelial cells, tissuemacrophages, and mast cells) and newly recruited inflammatory cells(such as monocytes, lymphocytes, neutrophils, and eosinophils).

Isolated: An “isolated” biological component (such as a nucleic acid orprotein) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e. other chromosomaland extrachromosomal DNA and RNA, and proteins. Nucleic acids andproteins that have been “isolated” thus include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cells, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes. Monoclonal antibodies are produced by methods known tothose of skill in the art, for instance by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells.

Natural killer (NK) cell: These are large, usually granular, non-T,non-B lymphocytes, which kill certain tumor cells. NK cells areimportant in innate immunity to viruses and other intracellularpathogens, as well as in antibody-dependent cell-mediated cytotoxicity(ADCC).

Neoplasm: An abnormal mass of tissue that results from excessive celldivision hat is uncontrolled and progressive, also called a tumor.Neoplasms can be begin (neither infiltrative nor cancerous) or malignant(invasive).

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least 6 nucleotides, for example at least 15, 25, 50,75, 100 or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for an agent to interact with a cell. “Contacting”includes incubating an agent in solid or in liquid form with a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of adenosine receptor inhibitorsand/or inhibitors of extracellular adenosine.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g. glycosylation or phosphorylation).

Preventing or treating a disease: “Preventing” a disease refers toinhibiting or decreasing the full development of a disease, for examplein a person who is known to have a predisposition to a disease. Anexample of a person with a known predisposition is someone with ahistory of diabetes in the family, or who has been exposed to factorsthat predispose the subject to a condition, such as lupus or rheumatoidarthritis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop.

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on a nucleic acid sequence. A probe includes an isolatednucleic acid attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g. in Sambrook et al.(In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) andAusubel et al. (In Current Protocols in Molecular Biology, Greene Publ.Assoc. and Wiley-Intersciences, 1992).

Primers are short nucleic acid molecules, such as DNA oligonucleotidesat least 10 nucleotides in length, such as about at least 12, 15, 17,20, or 25 nucleotides in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, e.g. bythe polymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (In Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1998), and Inniset al. (PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990). PCR primer pairs can be derivedfrom a known sequence, for example, by using computer programs intendedfor that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primerof 30 consecutive nucleotides of a adenosine receptor encodingnucleotide will anneal to a target sequence, such as another nucleicacid encoding an adenosine receptor, with a higher specificity than acorresponding primer of only 15 nucleotides. Thus, in order to obtaingreater specificity, probes and primers can be selected that include atleast 17, 20, 23, 25, 30, 35, 40, 45, 50 or more consecutive nucleotidesof nucleotide sequence of interest.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptide ornucleic acid preparation is one in which the peptide or nucleic acid ismore enriched than the peptide or nucleic acid is in its naturalenvironment within a cell. For example, a preparation is purified suchthat the protein or nucleic acid represents at least 50%, such as atleast 70%, of the total peptide or nucleic acid content of thepreparation.

Receptor: A molecular structure within a cell or on the surface of acell, characterized by selective binding of a specific substance and aspecific physiological effect that accompanies the binding, for example,cell surface receptors for peptide hormones, neurotransmitters,immunoglobulins, small molecules, and cytoplasmic receptors for steroidhormones. An adenosine receptor is a cell surface receptor foradenosine, and includes, but is not limited to, A2 and A3 receptors.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g. by genetic engineering techniques. Similarly, arecombinant protein is one encoded for by a recombinant nucleic acidmolecule.

Ribozyme: Synthetic RNA molecules that possess highly specificendoribonuclease activity. The production and use of ribozymes aredisclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No. 5,543,508to Haselhoff. The inclusion of ribozyme sequences within antisense RNAscan be used to confer RNA cleaving activity on the antisense RNA, suchthat endogenous mRNA molecules that bind to the antisense RNA arecleaved, which in turn leads to an enhanced antisense inhibition ofendogenous gene expression.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an antibody or antibody fragment-specific bindingagent binds substantially only the defined antibody or antibodyfragment, or an antibody region within a protein, such as a fusionprotein. As used herein, the term “adenosine receptor specific bindingagent,” includes anti-adenosine receptor antibodies (and functionalantibody fragments thereof) and other agents (such as potentialtherapeutic agents) that bind substantially only to adenosine receptors.

Antibodies can be produced using standard molecular procedures describedin a number of texts, including Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). The determination that aparticular agent binds substantially only to the target protein orpeptide can readily be made by using or adapting routine procedures. Onesuitable in vitro assay makes use of the Western blotting procedure(Harlow and Lane, Antibodies, A Laboratory Manual, CSHL, New York,1988).

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bindto adenosine receptor are adenosine receptor-specific binding agents.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

T Cell: A white blood cell involved in the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another example, a CD8 cell is asuppressor T cell.

Target sequence: A portion of ssDNA, dsDNA or RNA that, uponhybridization to a therapeutically effective oligonucleotide oroligonucleotide analog, results in the inhibition of gene expression,such as adenosine receptor gene expression. An antisense or a sensemolecule can be used to target a portion of dsDNA, since both willinterfere with the expression of that portion of the dsDNA. Theantisense molecule can bind to the plus strand, and the sense moleculecan bind to the minus strand. Thus, target sequences can be ssDNA,dsDNA, and RNA.

Therapeutically effective amount: A quantity of an agent or compositionsufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to increase activity of animmune cell and/or enhance an immune response in a subject. In oneexample, it is an amount that will inhibit viral, fungal, or bacterialreplication or to measurably alter outward symptoms of the viral,fungal, or bacterial infection. In another example, it is an amount thatwill decrease or prevent further tumor growth. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations (for example, in lymphocytes) that has been shown toachieve in vitro inhibition of viral replication or reduction of tumorcells.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, for example a dosesufficient to reduce the volume or size of a tumor. In another example,it is an amount which is capable of relieving symptoms caused by adisease, such as pain or swelling.

Therapeutically effective adenosine receptor oligonucleotides andoligonucleotide analogs: Characterized by their ability to inhibit ordecrease expression of one or more adenosine receptors. As describedbelow, complete inhibition is not necessary for therapeuticeffectiveness. Therapeutically effective oligonucleotides arecharacterized by their ability to inhibit or decrease the expression ofone or more adenosine receptors. Inhibition is a reduction in adenosinereceptor expression observed when compared to adenosine receptorproduction in the absence of the oligonucleotide or oligonucleotideanalog. For example, an oligonucleotides may be capable of inhibitingthe expression of adenosine receptors by at least 15%, 30%, 40%, 50%,60%, or 70%, or more, and still be considered to be therapeuticallyeffective.

Therapeutically effective oligonucleotides and oligonucleotide analogsare additionally characterized by being sufficiently complementary toadenosine receptor-encoding nucleic acid sequences. As described herein,sufficient complementary means that the therapeutically effectiveoligonucleotide or oligonucleotide analog can specifically disrupt theexpression of adenosine receptors, and not significantly alter theexpression of genes other than adenosine receptors.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes stablyreplicated by the cell, either by incorporation of the nucleic acid intothe cellular genome, or by episomal replication. As used herein, theterm transformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Treatment: Refers to both prophylactic inhibition of initial infection,and therapeutic interventions to alter the natural course of anuntreated disease process, such as infection with a virus.

Tumor: An abnormal mass of tissue that results from excessive celldivision that is uncontrolled and progressive, also called a neoplasm.Tumors can be benign (neither infiltrative nor cancerous) or malignant(invasive).

Vaccine: A dead or attenuated (non-pathogenic) form of a pathogen, or anantigen isolated from a pathogen, administered to a subject to induceadaptive immunity to the pathogen.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more selectablemarker genes and other genetic elements known in the art. The term“vector” includes viral vectors, such as adenoviruses, adeno-associatedviruses, vaccinia, and retroviruses vectors.

The Stop Mechanism of Inflammation

It is disclosed herein that the adenosine receptor is a physiological“stop” mechanism for inflammation in vivo, and as such, extracellularadenosine and adenosine receptors (such as A2a, A2b, and A3) arepharmacological and genetic targets for affecting inflammation andthereby altering the immune response. Inhibition or reduction ofextracellular adenosine or the adenosine receptor through the use aninhibitor of extracellular adenosine (such as an agent that prevents theformation of, degrades, renders inactive, and/or decreases extracellularadenosine), and/or an adenosine receptor inhibitor (such as an adenosinereceptor antagonist), as disclosed herein, is of use in generating animmune response, such as a macrophage, neutrophil, granulocyte,dendritic cell, T- and/or B cell-mediated response. In addition, aninhibitor of extracellular adenosine and adenosine receptor inhibitorsare of use in promoting acute or chronic inflammation. Inhibitors of theGs protein mediated cAMP dependent intracellular pathway and inhibitorsof the adenosine receptor-triggered G_(i) protein mediated intracellularpathways, can also be used to increase acute and chronic inflammation.

Inhibitors of: Adenosine Receptors, the Intracelluar cAMP DependentPathway and Extracellular Adenosine

Disclosed herein are methods for increasing an immune response orinflammation, and methods for targeted tissue damage, by contacting animmune cell or administering to a subject, one or more agents thatinhibit extracellular adenosine or adenosine receptor inhibitors, suchas adenosine receptor antagonists. A summary is provided in FIG. 16.

An agent that inhibits extracellular adenosine includes agents thatrender extracellular adenosine non-functional (or decrease suchfunction), such as a substance that modifies the structure of adenosineto nullify the ability of adenosine to signal through adenosinereceptors. This can be, for example, an enzyme (e.g. adenosinedeaminase) or another catalytic molecule that selectively binds anddestroys the adenosine, thereby abolishing or significantly decreasingthe ability of endogenously formed adenosine to signal through adenosinereceptors and terminate inflammation. One agent that degradesextracellular adenosine is ADA-PEG, polyethylene glycol-modified ADAthat has been used in treatment of patients with ADA SCID (Hershfield,Hum Mutat. 5:107, 1995). In another example, an agent that inhibitsextracellular adenosine includes agents that prevent or decreaseformation of extracellular adenosine, and/or prevent or decrease theaccumulation of extracellular adenosine.

Adenosine receptor inhibits include adenosine receptor antagonists. Anantagonist is any substance that tends to nullify the action of another,as an agent that binds to a cell receptor without eliciting a biologicalresponse. In one example, the antagonist is a chemical compound that isan antagonist for an adenosine receptor, such as the A2a, A2b, or A3receptor. In another example, the antagonist is a peptide, or apepidomimetic, that binds the adenosine receptor but does not trigger aG1 protein dependent intracellular pathway. Suitable antagonists aredescribed in U.S. Pat. Nos. 5,565,566; 5,545,627, 5,981,524; 5,861,405;6,066,642; 6,326,390; 5,670,501; 6,117,998; 6,232,297; 5,786,360;5,424,297; 6,313,131, 5,504,090; and 6,322,771.

In another example, the antagonist is an antisense molecule or catalyticnucleic acid molecule (e.g. a ribozyme) that specifically binds mRNAencoding an adenosine receptor. In specific, non-limiting examples, theantisense molecule or catalytic nucleic acid molecule binds A2a, A2b, orA3. In a further example, an antisense molecule or catalytic nucleicacid targets biochemical pathways downstream of the adenosine receptor.For example, the antisense molecule or catalytic nucleic acid caninhibit an enzyme involved in the Gs protein- or Gi protein-dependentintracellular pathway.

Adenosine receptor protein expression in a host cell can be reduced byintroducing into cells an antisense construct or another geneticsequence-targeting agent, based on an adenosine receptor locus, e.g. theadenosine receptor A1, A2a, A2b, or A3 locus (e.g. Genbank accessionnumbers L22214, AH003248, NM000676, and AH003597, respectively). Anantisense construct includes the reverse complement of the adenosinereceptor cDNA coding sequence, the adenosine receptor cDNA or genesequence or flanking regions thereof. For antisense suppression, anucleotide sequence from the adenosine receptor locus (e.g. all or aportion of the adenosine receptor cDNA or gene or the reverse complementthereof) is arranged in reverse orientation relative to the promotersequence in a vector. The vector is then introduced into a cell ofinterest. Where the reverse complement of the reported sequences is usedto suppress expression of proteins from the adenosine receptor locus,the sense strand of the disclosed adenosine or adenosine receptor locusor cDNA is inserted into the antisense construct. Without being bound bytheory, it is believed that antisense RNA molecules bind to theendogenous mRNA molecules and thereby inhibit translation of theendogenous mRNA.

For suppression of an adenosine receptor gene, transcription of anantisense construct results in the production of RNA molecules that arethe reverse complement of mRNA molecules transcribed from the endogenousadenosine receptor gene in the cell. The introduced sequence need not bethe full-length human adenosine receptor cDNA or gene or reversecomplement thereof, and need not be exactly homologous to the equivalentsequence found in the cell type to be transformed. Generally, however,where the introduced sequence is of shorter length, a higher degree ofhomology to the native adenosine or adenosine receptor locus sequence isneeded for effective antisense suppression. In one example, theintroduced antisense sequence in the vector is at least 10, such as atleast 30 nucleotides in length. Improved antisense suppression istypically observed as the length of the antisense sequence increases.Shorter polynucleotide (oligonucleotides) can conveniently be producedsynthetically as well as in vivo. In specific aspects, theoligonucleotide is at least 10 nucleotides, at least 15 nucleotides, atleast 30, at least 100 nucleotides, or at least 200 nucleotides.

To inhibit the translation of the target RNA molecule, such as anadenosine receptor, the antisense molecule will ideally persist in thecell for a sufficient period to contact the target RNA. However, cellscontains enzymes and other components that cause polynucleotides (suchas an antisense molecule) to degrade. The antisense molecule can beengineered such that it is not degraded in the cell. This can be done,for example, by substituting the normally occurring phosphodiesterlinkage which connects the individual bases of the antisense moleculewith modified linkages. These modified linkages can, for example, be aphosphorothioate, methylphosphonate, phosphodithioate, orphosphoselenate. Furthermore, a single antisense molecule can containmultiple substitutions in various combinations.

The antisense molecule can also be designed to contain different sugarmolecules. For example the molecule can contain the sugars ribose,deoxyribose or mixtures thereof, which are linked to a base. The basesgive rise to the molecules' ability to bind complementarily to thetarget RNA. Complementary binding occurs when the base of one moleculeforms a hydrogen bond with another molecule. Normally the base adenine(A) is complementary to thymidine (T) and uracil (U), while cytosine (C)is complementary to guanine (G). Therefore, the sequence ATCG of theantisense molecule will bond to TAGC of the target RNA. Additionally,the antisense molecule does not have to be 100% complementary to thetarget RNA to be effective.

The oligonucleotides can be DNA or RNA, or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, and can include other appendinggroups such as peptides, or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., PNAS USA 86:6553-6, 1989;Lemaitre et al., PNAS USA 84:648-52, 1987; PCT Publication No. WO88/09810) or blood-brain barrier (see, e.g., PCT Publication No. WO89/10134), hybridization triggered cleavage agents (see, e.g., Krol etal., BioTechniques 6:958-76, 1988) or intercalating agents (see, e.g.,Zon, Pharm. Res. 5:539-49, 1988).

In a particular example, an adenosine receptor antisense polynucleotideis provided, for example as a single-stranded DNA. Such a polynucleotidecan include a sequence antisense to a sequence encoding an A1, A2a, A2b,or A3 receptor. The oligonucleotide can be modified at any position onits structure with substituents generally known in the art. For example,a modified base moiety can be 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N˜6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

In another example, the polynucleotide includes at least one modifiedsugar moiety such as arabinose, 2-fluoroarabinose, xylose, and hexose,or a modified component of the phosphate backbone, such asphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, or a formacetal or analog thereof.

The antisense polynucleotide can be conjugated to another molecule, forexample a peptide, hybridization triggered cross-linking agent,transport agent, or hybridization-triggered cleavage agent. A targetingmoiety can also be included that enhances uptake of the molecule bytumor cells. The targeting moiety can be a specific binding molecule,such as an antibody or fragment thereof that recognizes a moleculepresent on the surface of the tumor cell.

Suppression of endogenous adenosine receptor locus expression can alsobe achieved using catalytic nucleic acids such as ribozymes. Ribozymesare synthetic RNA molecules that possess highly specificendoribonuclease activity. The production and use of ribozymes aredisclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No. 5,543,508to Haselhoff. Ribozymes can be synthesized and administered to a cell ora subject, or can be encoded on an expression vector, from which theribozyme is synthesized in the targeted cell (as in PCT publication WO9523225, and Beigelman et al. Nucl. Acids Res. 23:4434-42, 1995).Examples of oligonucleotides with catalytic activity are described in WO9506764, WO 9011364, and Sarver et al. (Science 247:1222-5, 1990). Theinclusion of ribozyme sequences within antisense RNAs can be used toconfer RNA cleaving activity on the antisense RNA, such that endogenousmRNA molecules that bind to the antisense RNA are cleaved, which in turnleads to an enhanced antisense inhibition of endogenous gene expression.

In addition, dominant negative mutant forms of an adenosine receptor canbe used to block endogenous adenosine receptor activity. In thisexample, a nucleic acid encoding a dominant negative mutant form of anadenosine receptor is operably linked to a promoter. In one specific,non-limiting example, the promoter is an inducible promoter. A vectorcontaining the promoter and the nucleic acid encoding the dominantnegative adenosine receptor is than introduced into a cell.

In another example, local tissue accumulation of extracellular adenosineis inhibited using a preparation of adenosine deaminase (ADA). This canbe, for example, an enzyme, adenosine deaminase or a ribozyme, oranother catalytic molecule that selectively binds and destroysadenosine, thereby abolishing, or substantially decreasing, the abilityof endogenously-formed adenosine to signal through adenosine receptorsand terminate inflammation.

The propagation of adenosine receptor-triggered intracellular signalingcascade can also be affected by the use of specific inhibitors ofenzymes and proteins that are involved in regulation of synthesis and/orsecretion of pro-inflammatory molecules, including modulators of nucleartranscription factors.

Suppression of adenosine receptor expression or expression of the Gsprotein- or Gi protein-dependent intracellular pathway, or the cAMPdependent intracellular pathway, are also used to increase/enhanceinflammation or the immune response in a variety of situations (seebelow).

Increasing an Immune Response

Methods are disclosed herein to enhance and prolong the pro-inflammatoryresponse by blocking the natural, extracellular adenosine-dependent,endogenous anti-inflammatory processes in vivo using an adenosinereceptor inhibitor and/or an inhibitor of extracellular adenosine. Inone example, the adenosine receptor is A2a, A2b, or the A3 receptor.

Also disclosed herein is a method for increasing an activity of animmune cell. Immune cells include, but are not limited to: leukocytes(i.e. neutrophils, eosinophils, lymphocytes, monocytes, basophils,macrophages, B cells, T cells, dendritic cells, and mast cells), as wellas other types of pro-inflammatory cytokine-producing cells. In anotherexample, the immune cell is a macrophage. In yet another example, theimmune cell is an antigen-presenting dendritic cell. In a furtherembodiment, the immune cell is a natural killer cell. In an additionalexample, the immune cell is a granulocyte. The immune cell activity canbe increased either in vivo or in vitro. In one example, this methodincludes targeting adenosine receptors on any other cell that producespro-inflammatory cytokines/molecules, including those that are notconsidered “classical immune cells.”

Thus, in one specific, non-limiting example the cell is a B cell, andsecretion of an immunoglobulin (e.g. IgG or IgM) is increased. Inanother specific, non-limiting example the cell is a T cell and theactivity is secretion of a cytokine (e.g. IL-2 or IL-4) is increased.Similarly, in another embodiment the cell is either a helper T cell or acytotoxic T cell, and either the helper T cell functions or thecytotoxic T cell functions are increased. Without being bound by theory,cytotoxic T cell activities are increased due to longer expression oflethal hit delivering Fas Ligand molecules or due to better traffickingof immune cells toward the targeted tissue in vivo. Without being boundby theory, T helper cell activities are enhanced because of prolongedsecretion of cytokines.

The method includes contacting the immune cell with an adenosinereceptor inhibitor, such as an adenosine receptor antagonist, or aninhibitor of extracellular adenosine, thereby increasing the activity ofthe immune cell. The immune cell can be involved in an acute immuneresponse or in a chronic immune response.

One of skill in the art can readily identify methods of use inidentifying an increased activity of an immune cell. For example,secretion of cytokines can be measured by ELISA or PCR-based assays orin biological assays. In one example, the increase in activity ismeasured as compared to a control. Suitable controls include an immunecell not contacted with an adenosine antagonist, or a standard value.

A method is disclosed herein for enhancing an immune response in asubject. The method includes administering to the subject atherapeutically effective dose of an adenosine receptor inhibitor and/oran inhibitor of extracellular adenosine, to enhance the immune response.In one example, the immune response is a macrophage/monocyte or B cellresponse. In another example the immune response is a T cell response.

A method is provided herein for improving a T cell mediated immuneresponse. The method includes the administration of an adenosinereceptor inhibitor and/or an inhibitor of extracellular adenosine, to asubject. In one embodiment, the subject is an immunosuppressed subject,such as a subject infected with an immunodeficiency virus (e.g. HIV-1 orHIV-2). The administration of the adenosine receptor inhibitor and/or aninhibitor of extracellular adenosine results in an increase in a desiredimmune response and/or prolonged secretion of a cytokine of interest. Inanother example, the subject is infected with a pathogen such as abacterial, viral, or fungal pathogen. Adenosine receptor inhibitorsand/or inhibitors of extracellular adenosine are administered tofacilitate pathogen destruction in the subject. In one example, thesubject is immunosuppressed. Immune deficiencies (e.g. deficiencies ofone or more type of immune cells, or of one or more immunologicalfactors) associated with immune deficiency diseases, immune suppressivemedical treatment, acute and/or chronic infection, and aging can betreated using the methods described herein. A general overview ofimmunosuppressive conditions and diseases can be found in Harrisons“Principles of Internal Medicine,” 14^(th) Edition, McGraw-Hill, 1998,and particularly in chapter 86 (Principles of Cancer Therapy), chapter307 (Primary Immune Deficiency Diseases), and chapter 308 (HumanImmunodeficiency Virus Diseases).

Many medical treatments can impair the immune system. Corticosteroids,for example, can reduce cell-mediated immunity. The predominant toxicityassociated with cancer therapies (e.g. chemotherapy and radiotherapy) isdestruction of proliferating cells, such as hematopoietic cells,responsible for maintenance of the immune and blood systems Likewise,immune suppression and depletion of the immune system is required forbone marrow transplantation, in which immune cells are eliminated andsubsequently replaced with transplanted cells. Certain knownimmunostimulants (e.g. erythropoietin and colony stimulating factorssuch as G-CSF, which is sometimes marketed under the name “Neupogen,”U.S. Pat. No. 5,536,495) have been used previously to treat certain ofthese conditions by stimulating regeneration of the immune cells. Theimmunostimulatory compounds and mixtures of the disclosure can be usedto stimulate the immune systems of patients suffering from medicaltreatment or iatrogenically induced immune suppression, including thosewho have undergone bone marrow transplants, chemotherapy, and/orradiotherapy.

Other conditions are known in which the immune system is compromised orsuppressed. For example, activation of the immune system (viastimulation of T cell production) by adenosine receptor antagonisttreatment can also be beneficial in aging subjects, in whom immunefunction is often compromised. Similarly, other conditions are known inwhich the immune response is abnormal or undesirable. Any of theseconditions would also benefit from the methods disclosed herein, orapplication of the described compositions. In general, the need fortreatment with one of the methods or compositions of this disclosure canbe determined by examining the immune status of a test subject, andcomparing this immune status to a control or average immune state (ahypothetical “normal” subject). For example, bone marrow biopsies orperipheral blood lymphocytes can be sampled to assess immune function.Indications of reduced immune function include leukopenia, for exampleneutrophenia or lymphopenia, or evidence of diminished white blood cellfunction. Where the test subject has a reduced immunity condition, suchas a reduction in a peripheral white blood cell count to below normal,for example 25% below normal, the immunostimulatory methods of thedisclosure should be considered as treatments to improve the immunesuppressed condition.

Also disclosed are methods that can be used to enhance NF-kB activity ina subject, by administering to a subject an adenosine receptor inhibitorand/or an inhibitor of extracellular adenosine. Enhancement of NF-kBactivity, promotes transcription of pro-inflammatory cytokines, such asIL-12p40 and TNF-α, thereby increasing an immune response.

Vaccines

A method is provided for increasing an immune response to an antigen byproviding an adjuvant activity. To increase an immune response to anantigen, the antigen is administered in conjunction with an inhibitor ofextracellular adenosine, an adenosine receptor inhibitor, an inhibitorof the intracellular cAMP dependent pathway, and/or an inhibitor ofintracellular Gi protein dependent pathway, which functions as anadjuvant.

In one example, a method for prolonging an immune response to a vaccineis provided. The method includes administering an adenosine receptorinhibitor in conjunction with the vaccine, such as an adenosine receptorantagonist.

Thus, disclosed herein is the use of an adenosine receptor inhibitor,such as an adenosine receptor antagonist, in adjuvant formulations.Methods for the stimulation of an immune response to a particularantigen are thus also within the scope of the disclosure. The hostanimals to which the adjuvant and adjuvant-containing vaccineformulations of the present disclosure are usefully administered includehuman as well as non-human mammals.

Typically, an antigen is employed in mixture with the adjuvant compoundsof the disclosure. Therapeutic adjuvant formulations are disclosedherein which, for example, include (i) at least one therapeuticallyeffective antigen or vaccine; and (ii) at least one adenosine receptorantagonist or adenosine degrading drug (e.g. ADA-PEG).

Such therapeutic composition can for example include at least oneantigenic agent such as (A) live, heat killed, or chemically attenuatedviruses, bacteria, mycoplasmas, fungi, and protozoa; (B) fragments,extracts, subunits, metabolites and recombinant constructs of (A); (C)fragments, subunits, metabolites and recombinant constructs of mammalianproteins and glycoproteins; (D) tumor-specific antigens; and (E) nucleicacid vaccines.

The therapeutic composition can therefore utilize any suitable antigenor vaccine component in combination with an adenosine receptorantagonist e.g. an antigenic agent, such as antigens from pathogenic andnon-pathogenic organisms, viruses, and fungi, in combination with anadjuvant compound of the disclosure.

As a further example, such therapeutic compositions can suitably includeproteins, peptides, antigens and vaccines which are pharmacologicallyactive for disease states and conditions such as smallpox, yellow fever,distemper, cholera, fowl pox, scarlet fever, diphtheria, tetanus,whooping cough, influenza, rabies, mumps, measles, foot and mouthdisease, and poliomyelitis. In the resulting vaccine formulation,comprising (i) an antigen, and (ii) at least one adenosine receptorinhibitor or and inhibitor of extracellular adenosine, the antigen andadjuvant compound are each present in an amount effective to elicit animmune response when the formulation is administered to a host animal,embryo, or ovum vaccinated therewith (see below).

Tumor Treatment

The importance of lymphoid cells in tumor immunity has been repeatedlyshown. A cell-mediated host response to tumors includes the concept ofimmunologic surveillance, by which cellular mechanisms associated withcell-mediated immunity destroy newly transformed tumor cells afterrecognizing tumor-associated antigens (antigens associated with tumorcells that are not apparent on normal cells). This is analogous to theprocess of rejection of transplanted tissues from a non-identical donor.In humans, the growth of tumor nodules has been inhibited in vivo bymixing suspensions of a patient's peripheral blood lymphocytes and oftumor cells, suggesting a cell-mediated reaction to the tumor. In vitrostudies have shown that lymphoid cells from patients with certainneoplasms show cytotoxicity against corresponding human tumor cells inculture. These cytotoxic cells, which are generally T-cells, have beenfound with neuroblastoma, malignant melanomas, sarcomas, and carcinomasof the colon, breast, cervix, endometrium, ovary, testis, nasopharynx,and kidney. Humoral antibodies that react with tumor cells in vitro havealso been produced in response to a variety of animal tumors induced bychemical carcinogens or viruses. Hybridoma technology in vitro permitsthe detection and production of monoclonal anti-tumor antibodiesdirected against a variety of animal and human neoplasms.Antibody-mediated protection against tumor growth in vivo has beendemonstrable in both leukemias and lymphomas.

A method is provided herein to increase inflammatory actions of immunecells including tumor-infiltrating lymphocytes, and in some embodiments,to additionally promote the recruitment of other immune cells withanti-tumor activity to improve the destruction of the tumor (such asreducing the size or volume of the tumor). A method is provided toimprove both natural anti-cancer immune response and adaptiveimmunotherapy of cancer by immune cells that recognize tumor-associatedantigens on the tumor cell surface. In one example, a first agent isadministered to a subject that has an affinity (tropism) for tumorcells. A second agent that is an adenosine receptor inhibitor (such asadenosine receptor antagonist) and/or an inhibitor of extracellularadenosine, is administered to the subject to promote the immune responseagainst the tumor. Without being bound by theory, the first agentselectively accumulates in the tumor due to tropism for the to tumorcells or the local environment. The first agent initiates the death ofsome low proportion of tumor cells due to its own cytotoxicity againsttumor cells.

In one example, the first agent induces cell death in the tumor cells.In an additional example, the first agent is a chemotherapeutic agent.In yet another embodiment, the first agent initiates an immune responsedirected against the tumor cells. In one example, the second agent is agenetic targeting agent used to mutate an adenosine receptor such thatthe receptor does not bind adenosine, or does not activate thebiochemical pathway triggered by the adenosine receptor. It is shownherein that by triggering low levels of inflammation in targeted tissues(e.g. tumors) with a first agent, in addition to complementaryinactivation of adenosine receptors or decreasing extracellularadenosine using genetic or pharmacological techniques, results indestruction of the tissue (e.g tumor).

In one example, the first agent is an immunotoxin that accumulates inthe tumor due to their selective interactions with tumor-specificantigens. These reagents cause direct destruction of tumor cells,although destruction of the tumor is not complete. Without being boundby theory, the death of a portion of the tumor cells creates aninflammatory environment within the tumor and activates tumorinfiltrating immune cells (macrophages and T cells). The naturalinhibitory pathway which would prematurely terminate this anti-tumoractivity will be then interrupted by the adenosine receptor inhibitor(such as adenosine receptor antagonist) or an inhibitor of extracellularadenosine (such as an extracellular adenosine degrading or disruptingagent). Thus, administration of a inhibitor of an adenosine receptorand/or an inhibitor of extracellular adenosine, exacerbates tumor celldeath.

In another example, the first compound initiates the anti-tumor processin vivo. A bi-functional immune cell activating reagent is coupled to anantibody that binds a tumor specific antigen and to a T cell ormacrophage-activating ligand (e.g. anti-T cell receptor mAb or Tcell-like receptor ligand, respectively). Without being bound by theorythe first agent accumulates in the tumor due to its selectiveinteractions with tumor-specific antigens. The first agent also directsactivation of tumor infiltrating immune cells, which destroys tumorcells. This activation of immune cells and tumor cells death willcreates an inflammatory environment within the tumor and also activatestumor infiltrating immune cells (e.g. macrophages and T cells). Thesecond agent is an adenosine receptor inhibitor (such as an adenosinereceptor antagonist) or an inhibitor of extracellular adenosine thatexacerbates tumor cell death.

In another embodiment, the first agent initiates an anti-tumor processin vivo is a population of T cells that are specific for tumor antigens,alone or in combination with other ligands that enhance antitumoractivity of T cells (e.g. CTLA-4 ligand; Kuhns et al., Proc. Natl. Acad.Sci. USA 97:12711, 2001) or in combination with the removal of CD25⁺regulatory T cells. Depletion of either of these two immunoregulatorymechanisms improves anti-tumor CTL activity (Sutmuller et al., J. Exp.Med. 94:823-32, 2001). Without being bound by theory, this activation ofimmune cells and tumor cells death creates an inflammatory environmentwithin the tumor and activates tumor infiltrating immune cells(macrophages and T cells). In this example, the second agent is not anadenosine receptor antagonist or adenosine degrading agent. Instead, itis the process of preparing anti-tumor immune cells under conditionsthat lead to the loss of (or reduction of) adenosine receptors, andthereby renders these cells uninhabitable by tumor-associated adenosine.This process can include additional conditions, such as hypoxicincubators to increase endogeneous adenosine formation in cell cultures,or addition of adenosine analogs to provide selective negative pressureto prevent or decrease expansion of adenosine receptor-expressing immunecells.

In an additional embodiment, the first agent is a cytotoxic compoundthat accumulates in tumor because of differences between tumor andnormal tissue environment (e.g. differences in growth rate, redoxpotential or oxygen tension (hypoxia) or other chemical differences).Without being bound by theory, this compound causes tumor cell death andcreates an inflammatory environment within the tumor and furtheractivates tumor infiltrating immune cells (macrophages and T cells). Thesecond agent is an adenosine receptor inhibitor (such as an adenosinereceptor antagonist) or an inhibitor of extracellular adenosine, thatexacerbates tumor cell death by preventing or decreasing theinactivation of anti-tumor cells by adenosine.

In yet another embodiment, the first agent is a compound thataccumulates in tumor cells and is cytotoxic due to the increasedproliferation of tumor cells Without being bound by theory, thiscompound causes tumor cell death and creates an inflammatory environmentwithin the tumor. Tumor infiltrating immune cells (macrophages and Tcells) are activated. The second agent is an adenosine receptorinhibitor (such as an adenosine receptor antagonist) or an inhibitor ofextracellular adenosine (such as an extracellular adenosine degradingagent) that exacerbates tumor cell death.

Pharmaceutical Compositions and Administration

Pharmaceutical compositions that include at least one adenosine receptorinhibitor, such as an adenosine receptor antagonist, and/or at least oneinhibitor of extracellular adenosine, can be formulated with anappropriate solid or liquid carrier, depending upon the particular modeof administration chosen. The pharmaceutically acceptable carriers andexcipients useful in this disclosure are conventional. For instance,parenteral formulations usually comprise injectable fluids that arepharmaceutically and physiologically acceptable fluid vehicles such aswater, physiological saline, other balanced salt solutions, aqueousdextrose, glycerol or the like. Excipients that can be included are, forinstance, other proteins, such as human serum albumin or plasmapreparations. If desired, the pharmaceutical composition to beadministered can also contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Other medicinal and pharmaceutical agents, for instance anotherimmunostimulant, also can be included. Immunostimulants include, but arenot limited to, IFA, COX-2 inhibitors, IL-12, saponins (e.g. QS-23), andN-acetyl-cysteine, for example.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical and oral formulations can be employed.Topical preparations can include eye drops, ointments, sprays and thelike. Oral formulations can be liquid (e.g. syrups, solutions orsuspensions), or solid (e.g. powders, pills, tablets, or capsules). Forsolid compositions, conventional non-toxic solid carriers can includepharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that comprise an adenosine receptorantagonist in some embodiments of the disclosure will be formulated inunit dosage form, suitable for individual administration of precisedosages. For example, one possible unit dosage can contain from about 1mg to about 1 g of adenosine receptor agonist or antagonist. The amountof active compound(s) administered will be dependent on the subjectbeing treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The compounds of this disclosure can be administered to humans or otheranimals on whose cells they are effective in various manners such astopically, orally, intravenously, intramuscularly, intraperitoneally,intranasally, transdermally, intradermally, intrathecally, andsubcutaneously. The particular mode of administration and the dosageregimen will be selected by the attending clinician, taking into accountthe particulars of the case (e.g. the subject, the disease, the diseasestate involved, and whether the treatment is prophylactic). Treatmentcan involve daily or multi-daily doses of compound(s) over a period of afew days to months, or even years.

A therapeutically effective amount of an adenosine receptor inhibitorand/or an inhibitor of extracellular adenosine, can be the amount ofadenosine receptor antagonist necessary to stimulate the immune systemof a subject. Specific immunostimulatory effects that can be caused byadenosine receptor antagonists as well as specific immunosuppressiveeffects that can be caused by adenosine receptor agonists are describedherein. In some embodiments, an immunostimulatory amount of an adenosinereceptor antagonist is an amount sufficient to stimulate an immuneresponse (for instance, any of the stimulatory responses discussedherein) without causing a substantial cytotoxic effect (e.g. withoutkilling more than 10% of cells in a sample).

An effective amount of an adenosine receptor inhibitor and/or aninhibitor of extracellular adenosine can be administered in a singledose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of an adenosine receptorinhibitor and/or an inhibitor of extracellular adenosine will bedependent on the specific agonist or antagonist applied, the subjectbeing treated, the severity and type of the affliction, and the mannerof administration of the therapeutic(s). For example, a therapeuticallyeffective amount of an adenosine receptor inhibitor and/or an inhibitorof extracellular adenosine can vary from about 0.1 mg/Kg body weight toabout 1 g/Kg body weight.

Site-specific administration of the disclosed compounds can be used, forinstance by applying an adenosine receptor antagonist to a precancerousregion, a region of tissue from which a neoplasm has been removed, or aregion suspected of being prone to neoplastic development.

The present disclosure also includes combinations of an adenosinereceptor inhibitor and/or an inhibitor of extracellular adenosine, withone or more other agents useful in the treatment of an immune-relateddisorder, condition, or disease. For example, the compounds of thisdisclosure can be administered in combination with effective doses ofother immunosuppressives, immunostimulants, anti-cancer agents,anti-inflammatories, anti-infectives, and/or vaccines. The term“administration in combination” or “co-administration” refers to bothconcurrent and sequential administration of the active agents. In oneexample, SEQ ID NO: 1 is co-administered with an adenosine receptorinhibitor and/or an inhibitor of extracellular adenosine. In anotherexample SEQ ID NO: 1 is administered before or after administration ofan adenosine receptor inhibitor and/or an inhibitor of extracellularadenosine.

Examples of agent that can be used in combination with the compounds ofthe disclosure are AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn),gamma interferon (Genentech), GM-CSF (Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F106528 (Genentech), TNF(Genentech), azathioprine, cyclophosphamide, chlorambucil, andmethotrexate. Treatment of a subject using the immunostimulatorycompositions of the disclosure can be indicated after (or while) thesubject has received an anti-proliferative or other cytotoxictherapeutic treatment. Examples of anti-proliferatives compounds includethe following: ifosamide, cisplatin, methotrexate, cytoxan, procarizine,etoposide, BCNU, vincristine, vinblastine, cyclophosphamide,gencitabine, 5-fluorouracie, paclitaxel, and doxorubicin.

In some examples, a subject is administered a cytotoxic treatment, thenmonitored for a period of time (usually in the range of days to weeks)to determine if the treatment leads to an immunosuppressive effect. Suchmonitoring can include monitoring peripheral blood for leukopenia orpancytopenia, and/or monitoring T cell function. A subject that displaysan immune suppression will be a candidate for treatment using thetherapeutic methods of the disclosed disclosure.

Methods for Screening for Adenosine Receptor Antagonists

Also disclose are methods for screening for adenosine receptorantagonist effects using a cell of the immune system. The methodincludes contacting an activated immune cell with the compound, andassaying the activity of the immune cell. An increase in activity or aprolonged period of activation of the immune cell indicates that thecompound is an adenosine receptor antagonist, which is efficient in anin vivo setting. Such methods can also be used to screen forimmunosuppressants and immunostimulants, wherein increased activity or aprolonged period of activation of the immune cell indicates that thecompound is an immunostimulant, and wherein decreased activity or areduction in the period of activation of the immune cell indicates thatthe compound is an immunosuppressant.

Among other uses, functional assays of receptor antagonist functionpermit optimization of the dosage amounts of each receptor agonist orantagonist effective in therapeutic uses. These assays can also be usedto test known adenosine receptor antagonists, as well as newlyidentified adenosine receptor antagonists or putative adenosine receptorantagonists for immunosuppressive or immunostimulatory bioactivity.Candidate agents can initially be screened for subsequent selection andtesting in one or more of the assays described herein.

Adenosine receptor antagonist immunostimulatory activity is the abilityof an adenosine receptor antagonist to enhance an immune response in animmune cell, an immune system, and/or more generally in a subject. Morespecifically, adenosine receptor antagonist-related immunostimulationincludes those effects that can be seen when an adenosine receptorantagonist is applied to an in vitro system using a dosage of less than2 pg/ml, and more particularly when less than 1 μg/ml (e.g., about 0.1μg/ml to as little as 0.003 μg/ml or less). In an in vivo system, theseeffects are seen at an application level of about 1 μg to about 5 μg ina 20 g mouse, or about 50 to about 250 μg/Kg body weight. Specificimmunostimulatory effects that can be involved include stimulation ofT-cell production, stimulation of interleukin production (e.g.production of IL-1 and/or IL-12 by macrophages), and activation ofnatural killer cells and/or macrophages.

Methods for examining adenosine receptor inhibitor-mediatedimmunostimulation include those disclosed herein, such as directmeasurement of the activation or proliferation of one or more immunecell types, or increased (or decreased) interleukin production (e.g.IL-1 or IL-12). Secondary effects of immune stimulation can also bemeasured as described herein, for instance by examining the formation oftumors, the relative rate of growth of a tumor, or tumor metastasis, orby resistance of an organism treated with the test compound to viral orother infection.

Example 1 Activation of A2a Receptors Reduces Concanavalin-A-InducedLiver Damage In Vivo

This example describes methods that were used to demonstrate thatpharmacological activation of A2a receptors by a selective A2a agonistprevents liver damage in a model of inflammatory liver injury.

Concanavalin A (Con-A)-induced liver injury in mice is mediated byT-cells, macrophages and pro-inflammatory cytokines TNF-α, IL-4 andIFN-γ, and represents a well-described in vivo inflammation model ofviral and autoimmune hepatitis. Five B6 wild type mice were injectedintravenously (i.v.) with 20 mg/kg Con A (type IV, Sigma, St. Louis,Mo.) in sterile PBS alone or co-injected intraperitoneally (i.p.) withCGS21680 (2 mg/kg), isoproterenol (100 mg/kg, Sigma, St. Louis, Mo.), orprostaglandin E₂ (PGE₂, 5 mg/kg, Sigma, St. Louis, Mo.) just before ConA treatment. The extent of liver damage and inflammation were quantifiedby measuring serum levels of liver enzyme alanine aminotransferase (ALT)and TNF-α at 1.5 hours, 4 hours, and 8 hours after Con-A injection.TNF-α was measured using an ELISA kit (R&D systems, Minneapolis, Minn.)according to manufacturer suggestions. Serum ALT activity was determinedusing a colorimetric assay kit (Sigma, St. Louis, Mo.). The datadisclosed herein are expressed as mean+/−s.e.m. Differences betweengroups were evaluated using Student's t-test.

As shown in FIG. 1A, pharmacological activation of severalcAMP-elevating G_(s)-coupled receptors prevented or reduced liverdamage. Similarly, activation of A2a, inhibited Con-A-inducedpro-inflammatory TNF-α accumulation in vivo. The A2a agonist, CGS21680,also inhibited secretion of IL-12 and IFN-γ by activated macrophages andT cells in vitro as measured using an ELISA kit (R&D systems,Minneapolis, Minn.) (FIG. 1B). Therefore, pharmacological activation ofA2a receptors or other G_(s)-protein-coupled receptors in vivo preventsor reduces Con-A-induced liver damage and pro-inflammatory TNF-αaccumulation.

Example 2 Effect of A2a Agonists and Antagonists on cAMP Levels in MouseLiver Mononuclear Cells In Vitro

This example describes methods that were used to demonstrate that A2areceptor signaling (cAMP accumulation) is decreased or even abolished inliver mononuclear cells and macrophages from A2aR^(−/−) mice but not incells from A2aR^(+/+) littermates.

Littermates or age-matched wild type (A2aR^(+/+)) and homozygous A2areceptor gene deficient mice (A2aR^(−/−)) were used in all experimentsfor better reproducibility of results. C57BL/6-background A2areceptor-deficient mice have been described previously (Chen et al., J.Neuroscience 19:9192-9200, 1999; Apasov et al., Br. J. Pharmacol.131:43-50, 2000; and Armstrong et al., Biochem. J. 354:123-130, 2001).The A2a receptor genotypes of mice were determined by Southern blotanalysis (Chen et al., J. Neuroscience 19:9192-200, 1999).

Stimulation of cells and measurement of cAMP levels were carried out asdescribed previously (Apasov et al., Br. J. Pharmacol. 131:43-50, 2000,herein incorporated by reference). Briefly, liver mononuclear cells wereisolated from parenchymal hepatocytes and cell debris using Percoll(Amersham Pharmacia Biotech, Uppsala, Sweden). The resulting livermononuclear cells (1×10⁵ cells/200 μl) were incubated at 37° C. for 30minutes in the presence of 10 μM CGS21680, 1 μM ZM241385 (Tocris,Ballwin, Mo.), 100 μM isoproterenol, 50 μM forskolin, or 1 μM PGE₂. ThecAMP levels were determined using a cAMP enzyme immunoassay kitaccording to the manufacturer's instructions (Amersham PharmaciaBiotech, Buckinghamshire, England, UK).

As shown in FIG. 2A, the A2aR antagonist, ZM241385, inhibited AGORA2aagonist (CGS21680)-induced cAMP increase in mononuclear cells fromA2aR^(+/+) mouse livers. By contrast, the A2aR agonist, CGS21680, didnot increase cAMP in mononuclear cells from A2aR^(−/−) mouse livers(FIG. 2B). Cells from A2aR^(−/−) mice retained the cAMP response toligands of other Gs protein coupled receptors (FIG. 2D). Therefore, A2areceptors can downregulate inflammation when activated by agonists.Therefore, there is a deficiency in cAMP-elevating receptors in A2adeficient mice. As a result, these mice can be used to establish therole of A2a receptors in inflammation in vivo.

Example 3 Accumulation of Inflammatory Cytokines and Liver Damage inA2a-Receptor-Deficient Mice

This example describes methods that were used to demonstrate that theabsence of functional A2a receptors results in increased inflammationand exacerbated tissue damage, in response to administration of Con-A.

A2a^(+/+) (n=5) and A2a receptor gene deficient (A2a^(−/−)) mice (n=5)were used. Mice were injected intravenously with a sub-optimal dose(12.5 mg/kg) of Con-A (see inset, FIG. 3A), and subsequently, serumlevels of ALT and cytokines were measured at 1, 6, 8, 24, and 48 hoursas described in EXAMPLE 1 (cytokines were measured using an ELISA kit(R&D systems) according to manufacturer suggestions).

As shown in FIG. 3A, a large increase in serum ALT levels in A2a^(−/−)mice as compared to A2a^(+/+) mice was observed following Con-Atreatment. Even a sub-optimal dose of inflammatory stimuli, Con-A (seeinset to FIG. 3A) resulted in the death of two out of five A2aR^(−/−)mice within 48 hours, while all A2aR^(+/+) controls survived. Low dosesof Con-A, which caused only minimal or no liver damage in controlA2aR^(+/+) mice, were sufficient to induce extensive inflammation andliver damage in A2aR^(−/−) mice, as evidenced accumulation of dead cellsand leukocytes using TdT apoptosis test and haematoxylin and eosin (H-E)stain.

The effect of TNF-α on liver injury in A2aR^(+/+) (n=5) and A2aR^(−/−)(n=5) mice was compared by injecting mice with a combination ofD-galactosamine (Sigma, St. Louis, Mo.) and TNF-α (PharMingen, SanDiego, Calif.). D-galactosamine (700 mg/kg) was injectedintraperitoneally 30 minutes before i.v. injection of recombinant mouseTNF-α (4-15 μg/kg). After six hours, mice were sacrificed and the liverdamage was evaluated by measuring serum ALT levels as described inEXAMPLE 1. As shown in FIG. 3B, deficiency in A2a receptors did notaffect the susceptibility of hepatocytes to in vivo damage by TNF-α.Therefore, differences between A2aR^(+/+) and A2aR^(−/−) mice were notexplained by increased susceptibility of A2aR^(−/−) hepatocytes toTNF-α, since TNF-α was equally efficient in directly destroyinghepatocytes in both A2aR^(−/−) and A2aR^(+/+) mice in vivo (FIG. 3B).Furthermore, excessive and prolonged pro-inflammatory TNF-α accumulationwas observed in the serum of A2aR^(−/−) mice compared to low orundetectable TNF-α levels in A2aR^(+/+) mice (FIG. 3B). IFN-γ was alsopresent at higher concentrations and for a greater duration inA2aR^(−/−) mice, although levels of IL-4 were not different betweenA2aR^(−/−) and A2aR^(+/+) mice. Inactivation of A2a receptors inA2aR^(+/±) wild type mice using the A2 receptor antagonist ZM241385,also increased inflammatory tissue damage in A2aR^(−/−) mice (FIG. 4).

In summary, other cAMP-triggering Gs-coupled receptors do notappreciably compensate for the lack of A2a receptors on immune cells ofA2aR^(−/−) mice, as demonstrated by the increased sensitivity ofA2aR^(−/−) mice to Con-A. Mice with genetically inactivated adenosinereceptors indeed lack functional adenosine receptors. A2aR^(−/−) micehave other fully functional receptors (e.g. prostaglandin orbeta-adrenergic receptors), which may function as naturaldown-regulators of immune response. These data demonstrate that A2areceptor gene deficient mice can be used to implicate A2a receptors asnon-redundant downregulators of immune response in vivo. These data alsodemonstrate that the signal transduction pathway leading to cAMPaccumulation in these mice is functional, thereby completely excludingany possibility that an artifactual mutation exists in A2aR genedeficient mice.

Example 4 Inactivation of A2a Receptors In Vivo Exacerbates Liver Damage

This example provides methods that were used to demonstrate thetissue-protecting properties of A2 adenosine receptors in other modelsof inflammatory liver injury and systemic inflammation. These resultsdemonstrate that targeted tissue damage can be achieved in vivo, forexample when the targeted tissue is a tumor, the first agent or drug isspecific immune cell (e.g. T cells or NK-T cells), a toxin (e.g. PEA) ora cytotoxic agent (e.g. carbon tetrachloride).

B6 mice (n=5) were injected with 12.5 mg/kg of Con-A alone or incombination with A2aR antagonist ZM241385 (2 mg/kg). PseudomonasExotoxin A (PEA, 100 μg/kg i.v., Sigma, St. Louis, Mo.) was also used toinduce liver injury as follows. Mice were injected with PEA alone (n=6)or in combination with an i.p. injection of ZM241385 (n=7) before and 12hours after the PEA injection. Carbon tetrachloride (CCl₄)-inducedhepatotoxicity was determined by injecting (i.p.) A2aR^(+/+) (n=7) andA2aR^(−/−) (n=7) mice with CCl₄ (0.5 ml/kg, Sigma) dissolved in oliveoil.

Subsequent to the injections, serum ALT measurements were made asdescribed in EXAMPLE 1, which indicate the extent of liver damage. Inaddition, histological evaluations and an analysis of tissue damage andapoptotic cells (the detection of apoptotic cells by in situ staining ofsingle strand breaks in nuclear DNA) were determined.

As shown in FIG. 4A, pharmacological inactivation of A2a receptors invivo using an adenosine receptor antagonist, exacerbates Con-A-inducedliver damage. Inactivation of A2 adenosine receptors in A2aR^(+/+) miceby antagonist ZM241385, exacerbated the T cell- and TNF-α-dependentacute hepatotoxicity of PEA (FIG. 4B). Increased liver injury was alsoobserved in A2aR^(−/−) mice during chemically (CCl₄)-induced acutehepatotoxicity (FIG. 4C).

Therefore, enhanced and prolonged accumulation of pro-inflammatorycytokines and exaggerated liver damage occurs in A2a-receptor-deficientmice as compared to wild-type mice. These data demonstrate thatadenosine receptors, such as A2a receptors, function in vivo asphysiological downregulators of immune response/inflammation andfunction as protectors from excessive tissue damage. Geneticinactivation of A2a receptors results in much stronger, longerinflammatory response to very low, sub-optimal doses of pro-inflammatorystimuli. This is evidenced by tissue damage and death of animals(virtually no tissue damage and only short duration of higher levels ofpro-inflammatory cytokines were detected in normal wild type littermateswhich express A2a receptors). Virtually no tissue damage was detected innormal wild type littermates given the same dose of pro-inflammatorystimuli, Con-A without the administration of the A2aR antagonist.

In summary, these results demonstrate that a targeted tissue (in thisexample it was liver, but other tumors can be targeted) can be reducedor destroyed by disengaging immunosuppressive “brakes” using two agents.The first agent is target tissue-specific, such as cytotoxic cells withtropism to a tumor, which can increase T-cell dependent tissue damage;such as an immunotoxin with tropism to a tumor (FIG. 4B, which canresult in immunotoxin-dependent targeted tissue damage; and such as atoxic chemical agent with tropism to the tumor (FIG. 4C), which canresult in chemotherapy-dependent targeted tissue damage. This agentinitiates non-observable or low intensity inflammation. The second agentinactivates or decreases hypoxia, extracellular adenosine, and/or thepresence of adenosine receptors. This second agent enhances theintensity and prolongs the duration of targeted tissue destruction.

Example 5 Enhanced Accumulation of Pro-inflammatory Cytokines and TissueDamage in Endotoxin-Treated A2a Receptor-Deficient Mice

This example describes methods further used to demonstrate the role ofA2a adenosine receptors in down-regulating pro-inflammatory cytokineaccumulation and tissue damage, using an in vivo septic shock modelfollowing subcutaneous and i.v. bacterial endotoxin (LPS) injection.

Lipopolysaccharide (LPS, E. coli 0111:B4; 3 mg/kg, Sigma) was injectedi.v. into A2aR^(−/−) (n=11) and A2aR^(+/+) (n=10) mice. Survival wasmonitored for 96-120 hours after LPS injection. Statistical analysisconfirmed the higher and faster mortality of mice without adenosinereceptors. As shown in FIG. 5, all A2a^(−/−) mice were dead by 48-72hours, whereas survival of A2a^(+/+) mice was 10-20% at 120 hours or 96hours.

Endotoxic shock in male A2aR^(−/−) mice and age-matched A2aR^(+/+) micewas induced by i.v. injection of 3 or 5 mg/kg LPS. Subsequently, at 1hour and 16 hours after injection, blood samples were obtained byretro-orbital bleeding. In another group of mice, LPS was injected (100μg/kg) into a dorsal air pouch, which was prepared using sterile airessentially as described in Levy et al. (Nat. Immunol. 2:612, 2001).Serum cytokine levels were determined at different times after LPSinjection using ELISA kits obtained from R&D systems according tomanufacturer's suggestions as follows: TNF-α and IL-6 levels weremeasured at 1 hour, and IL-12p40 and IL-1β levels at 3 hours. As shownin FIGS. 6A-D, absence of A2a adenosine receptors dramatically increasesthe level of pro-inflammatory cytokines in A2aR^(−/−) mice as comparedto wild-type mice after air pouch LPS injection (infected wound model).

Therefore, A2a adenosine receptors protect against death from septicshock, as mice lacking adenosine receptors (due to geneticinactivation), die faster (FIG. 5) and have higher levels of cytotoxicTNF-α (FIG. 6A) in response to bacterial endotoxin. These resultsdemonstrate that A2a adenosine receptors are the natural andnon-redundant “brakes” of inflammatory tissue damage.

Example 6 Enhanced Accumulation of Pro-Inflammatory Cytokines mRNA inA2aR^(−/−) Mice after Activation of Immune Cells

Mutant (A2aR^(−/−)) or wild-type mice were injected (i.p.) with 20 nmolCpG oligonucleotide (5′-T*C*CATGACGTTCCTG*A*T*G*C*T-3′, asterisk meansphosphorothioate. SEQ ID NO. 1). This toll-receptor activating CpG DNApreparation stimulates the immune system. After one hour, mRNA wasextracted from splenocytes and analyzed for cytokine gene expressionusing an RNase protection assay with commercial templates (mCK-2b andmCK-3b, Pharmingen, San Diego, Calif.).

As shown in FIG. 7, NF-kB transcribed cytokine mRNAs (such as TNF-α andIL-12p40) are dramatically increased in the absence of adenosinereceptors. Therefore, A2a adenosine receptors are involved indown-regulating pro-inflammatory cytokine mRNA accumulation includingIL-12 (which is important for the T cell response), during in vivoactivation of immune cells by CpG. This provides further evidence thattargeted inactivation of adenosine receptors can be used to enhance theimmune response. Since cytokine IL-12 is important in promoting T-celldependent immune response, these data demonstrate that genetic targetingthe “adenosine accumulation->adenosine->adenosine receptors->signaling”pathway in immune cells can enhance an immune response, which can beused as an immunoenhancer to improve vaccines.

Example 7 Adenosine Receptor Antagonists Increase Expression ofInflammatory Cytokines in CpG-Activated Immune Cells In Vivo

This example describes methods used to determine the role of adenosinereceptor antagonists on NF-KB activity and expression of inflammatorycytokines such as TNF and IL-12p40.

C57BL/6 mice were pretreated with ZM241385 (10 mg/kg i.p.) 15 minutesbefore administration of SEQ ID NO: 1, and the expression of cytokinemRNA in the spleen was compared with the mice treated with CpG alone,using the methods described in EXAMPLE 6.

As shown in FIG. 7, in the adenosine receptor mutant mice (adenosinereceptors genetically inactivated), there was higher mRNA expression ofNF-kB-regulated pro-inflammatory cytokines (TNFα, IL-12p40). Similarly,as shown in FIGS. 8A and 8B, higher expression of NF-kB-regulatedpro-inflammatory cytokines is observed in ZM241385-treated mice(adenosine receptors pharmacologically inactivated). Therefore,administering an adenosine receptor antagonist can be used to increaseor enhance the transcription of pro-inflammatory cytokines following CpGactivation, due to enhanced or increased NF-kB activity.

Example 8 Adenosine Receptors Decrease NF-kB Nuclear Translocation andCytokine mRNA Transcription by Inhibiting IKK-Mediated Phosphorylationof IkB

To further demonstrate the role of adenosine receptors on IKK-mediatedphosphorylation, which is necessary for NF-kB nuclear translocation,which is needed for expression of cytokines, the following methods wereused.

Mutant (A2aR^(−/−)) and wild-type mice were injected (i.v.) with CpG toactivate immune cells as described in EXAMPLE 6. One hour after theinjection, nuclear extracts from peritoneal macrophages were isolatedusing standard methods. Nuclear extracts were compared inelectrophoretic mobility shift assay (EMSA) for binding to specific DNAsequences according to routine EMSA methods, to determine the extent ofNF-kB translocation into the nucleus of macrophages.

As shown in FIG. 9, CpG-induced NF-kB translocation into the nucleus isincreased in the absence of adenosine A2a receptors. This demonstratesthat A2a receptors negatively regulate NF-kB translocation into thenucleus, and thereby its activity, in vivo.

To determine the role of IkB phosphorylation, C57BL/6 mice wereadministered (i.v.) 5 nMol of CpG (SEQ ID NO: 1) in the presence orabsence of the adenosine receptor agonist CGS21680 (2 mg/kg). After 20minutes, macrophages were isolated as described above, and subjected toWestern blot analysis using Ab that recognize IkB and Ab can distinguishthe phosphorylated form of IkB (IkB-P). As shown in FIG. 11A,phosphorylation of IkB was decreased or inhibited in the presence of theadenosine receptor agonist CGS21680, following immune stimulation withCpG. As shown in FIG. 11B, in control panels the parallel samples hadsimilar levels of IkB as shown in Western blots.

Therefore, NF-kB activity is regulated by adenosine receptors due to theinhibition of (or decrease of) phosphorylation of IkB by IKK. In theabsence of phosphorylation of IkB, NF-kB cannot translocate into thenucleus, and therefore NF-kB cannot induce mRNA expression ofinflammatory cytokines such as IL-12p40 and TNF-α. Enhancement of NF-kBtranscription factor nuclear translocation by inhibition of A2 adenosinereceptor-mediated signaling is explained by prevention of cAMP-inducedinhibition of phosphorylation of IkB by IKK.

As summarized in FIG. 11, the presence of active adenosine receptors,such as A2a, decreases or inhibits inflammation by blocking IKK-mediatedIkB phosphorylation, and NF-kB nuclear translocation, thereby inhibitingor decreasing mRNA expression of pro-inflammatory cytokines.

Example 9 Adenosine Receptor Antagonists Decrease Tumor Growth

To determine if a tumor self-protection mechanism could be defeated byreducing the presence of adenosine, the following methods were used.Because tumors are hypoxic, and hypoxia is conducive to adenosineaccumulation in the brain, heart, and in solid tumors, it is possiblethat adenosine inhibits or prevents anti-tumor immune cells (such asT-killer cells) from contacting the tumor, thus preventing the tumorfrom being acted upon by the anti-tumor cells. For example, the presenceof adenosine may inhibit or decrease the signaling of CTL chemokinesreceptors (which may result in a decreased attraction to a tumor and/ora decrease or inhibition of chemotaxis); inhibit or decrease motility ofCTLs; inhibit or decrease production of inflammatory cytokines by CTL;inhibit or decrease the formation of CTL/tumor conjugates; inhibit ordecrease FasL/granule exocytosis; and/or inhibit or decrease a lethalhit by CTL. As a result, if adenosine is blocked or reduced, thenanti-tumor cells may be more effective in reducing a tumor.

BALB/c mice were inoculated i.v. with CMS4 tumor cells(Methylcholanthrene-induced sarcoma, 2.5×10⁵ cells) on day zero toinduce the formation of lung tumors. Ten-days later, antigen-specificT-killer cells (CTL) cells (5×10⁵ or 1×10⁶ cells) were injected into themice (i.v.) in the presence or absence of an i.p. injection of ZM241385(10 mg/kg/day) or administered a relatively nonselective antagonist ofA2a and A2b receptors, 1,3,7-thrimethylxantine (caffeine, 0.1% w/v) viadrinking water to inactivate A2 Receptors on the CTL-cell surface, sinceit is through these receptors the tumor signals T-killer cells andthereby stop them from delivering the lethal “hit” to tumor cells. Thelung tumors were subsequently examined on days 17, day 18 and day 24, bysacrificing the mice and evaluating their lungs for the number and sizeof metastasis by visual inspection.

As shown in FIGS. 12A-C, administration of an adenosine receptorantagonist, such as ZM241385 or caffeine, greatly improves immunotherapyof cancer tumors, as evidenced by a decrease in the number of metastaticnodules in the lung. In contrast, CTL cells alone were not as capable ofefficiently reducing tumor metastasis.

To demonstrate that similar results are obtained with non-immunogeneictumors, such as breast tumors, the following methods were used. BALB/cmice were inoculated subcutaneously with 1×10⁵ non-immunogeneic 4T1breast tumor cells (American Type Culture Collection, Manassas, Va.,Catalog No. CRL-2539) on day zero to induce the formation of breasttumors. When injected into BALB/c mice, 4T1 cells spontaneously producehighly metastatic tumors that can metastasize to the lung, liver, lymphnodes and brain while the primary tumor is growing in situ. Seven daysafter tumor inoculation, mice were injected daily (i.p.) with ZM241385(10 mg/kg) or caffeine (20 mg/kg). Time-dependent changes of tumordiameter and tumor volume were subsequently calculated.

As shown in FIG. 13, adenosine receptor antagonists slow the growth ofnon-immunogeneic 4T1 breast tumor cells (s.c.), indicating thatadministration of an adenosine receptor antagonists to a subject havinga tumor can reduce the number and/or size of one or more tumors in thesubject. These results also indicate that adenosine receptor antagonistsreduce tumor growth by impairing angiogenesis.

Example 10 Adenosine Receptor Antagonists Decrease Tumor Size

This example describes methods that were used to improve anti-tumorvaccination by co-administering an adenosine receptor antagonist.

BALB/c mice (immunocompromised nude mice; or immunocompetent C57BL/6mice) were inoculated subcutaneously with B16 melanoma cells (AmericanType Culture Collection, Manassas, Va.), or B16-H2-Kd transfected tumorcells (increases the immunogenicity of the cells) on day zero to inducethe formation of melanomas. The treatment with adenosine receptorantagonists started 28 days after injection of tumor cells, when thetumor reached 7-9 mm in diameter.

As shown in FIG. 14, daily i.p. treatments with ZM241385 (0.2 mg/mouse)and caffeine (0.4 mg/mouse) resulted in tumor retardation that becamesignificant after 3-7 days of treatment. The delay in tumor growth wasgreater in immunocompetent C57BL/6 mice, and less so inimmunocompromised nude mice. However, as the tumor increases in size,both antagonists slow down tumor growth even in immunocompromisedanimals. This indicates that administration of adenosine receptorantagonists to a subject having a tumor can reduce the number and/orsize of one or more tumors in the subject. In addition, adenosinereceptor antagonists appear to improve both anti-tumor immunity andpreventing or decreasing angiogenesis.

Example 11 Adenosine Receptor Antagonists Improve Immune Response toSubcutaneous and Intra-Peritoneal Vaccination

This example describes methods used to determine if an immune responseto vaccines can be improved by co-administering an adenosine receptorantagonist, such as A2a and A3 antagonists, and whether any effects isaltered if the vaccine is delivered via different delivery routes.

TNP-KLH (100 μg, Sigma), a model antigen, was injected i.p. orsubcutaneously into the footpad of A2aR^(+/+) mice along with anadenosine receptor antagonist (about 1 mg/kg of theophylline (an A2aantagonist), ZM241385 (an A2a antagonist), or MRS 1220 (an A3antagonist, Sigma)). The antigen was prepared for injection as follows.DNP-KLH (1.0 mg/ml, Biosearch Technologies, Inc. catalog no. T-5060-5)was prepared in PBS (e.g. dissolve 4.0 mg of purified DNP-KLH in PBS andraise volume to 4.0 ml). Complete Freund's Adjuvant (2.0 ml, CFA; SigmaF-5881) was vortexed and mixed with 2.0 ml of the TNP-KLH solution at 4°C. The CFA/KLH mixture was drawn into a 3-ml glass syringe with a19-gauge needle. The syringe was attached to a double-ended locking hubconnector or a plastic 3-way stopcock. An empty 2-ml glass syringe wasattached to the other end and the mixture forced back and forth from oneend to the other repeatedly. When the mixture was white and homogeneous,the connector or stopcock was disconnected, a 25 gauge needle attached,and tested for emulsion by placing a drop on the surface of 50 ml ofcold water in a 100-ml beaker. The drop should hold together; if not,repeat mixing. 200 μl (100 μg) was injected intraperitoneally (i.p.)into each mouse.

Samples of blood were obtained by retro-orbital bleeding at 7, 14, and21 days after TNP-KLH and theophylline injection. Serum levels ofanti-TNP-specific IgG1, IgG2a, IgG2b, IgG3, and IgM were estimated usingan ELISA kit (Sigma) according to the manufacturer's instructions.

Serum levels of anti-TNP-specific IgG1 were markedly increased at sevendays in mice co-injected with TNP-KLH and ZM241385 or MRS1220, ascompared to control. IgG₂ was also improved on day 7. Therefore,blocking the endogenous anti-inflammatory pathway using adenosinereceptor antagonists improves immune response to vaccination (asevidenced by higher titers of antigen-specific immunoglobulin IgG1),when adenosine receptor antagonists are administered with a vaccine.

Similarly, serum levels of anti-TNP-specific IgG1 were markedlyincreased at seven days in mice co-injected i.p. (FIG. 15) orsubcutaneously with TNP-KLH and theophyllin, as compared to control.Therefore, co-administration of an adenosine receptor antagonist with avaccine potentiates the immune response to the vaccine, when vaccinationis intra-peritoneal or subcutaneous. Pharmacological inactivation ofadenosine receptors, such as A2a and A3, using antagonists results inhigher titers of antigen-specific immunoglobulins IgG₁, which suchantagonists are administered with a vaccine.

Example 12 Administration of Adenosine Receptor Antagonists withAnti-Cancer Agents

This example describes methods that can be used to facilitate thetreatment of cancer in a subject using one or more adenosine receptorantagonists, alone or in combination with anti-cancer agents. Thisprotocol serves as an example of such a treatment method, and is notlimiting. Those of skill in the art can modify the protocol to suit theneeds of the subject, and to optimize for the particular agents used.Subjects can, but need not, have received previous chemo- radio- or genetherapeutic treatments. Optimally, the patient will exhibit adequatebone marrow function (defined as peripheral absolute granulocyte countof >2,000/mm³ and platelet count of 100,000/mm³.

Adenosine receptor antagonists are administered orally or parenterallyin dosage unit formulations containing standard, well known non-toxicphysiologically acceptable carriers, adjuvants, and vehicles as desired.The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intra-arterial injection, or infusiontechniques. Adenosine receptor antagonists can be administered indosages of about 0.1 mg/kg to about 1 g/kg, depending on the antagonistused. For example, adenosine receptor antagonists can be administered toa subject at a dose of least 0.5 mg/kg of body weight, such as 3-10mg/kg. The adenosine receptor antagonists can be delivered to thepatient before, after or concurrently with the other anti-cancer agents.

A typical treatment course can include about six doses delivered over a7 to 21 day period. Alternatively, a treatment course can include dailydoses delivered over a 7 to 21 day period. Upon election by theclinician, the regimen can be continued six doses every three weeks oron a more frequent (daily, twice daily, four times a day, etc.) or lessfrequent (monthly, bimonthly, quarterly, etc.) basis. Of course, theseare only exemplary times for treatment, and the skilled practitionerwill readily recognize that many other time-courses are possible. Theadenosine receptor antagonists can be combined with any of a number ofconventional chemotherapeutic regimens.

Regional delivery of adenosine receptor antagonists is an efficientmethod for delivering a therapeutically effective dose to counteract theclinical disease. Likewise, the chemotherapeutic agents can be directedto a particular affected region. Alternatively, systemic delivery ofeither or both agents can be appropriate.

Clinical responses can be defined by an acceptable measure. For example,a complete response can be defined by the disappearance of allmeasurable disease for at least a month. A partial response can bedefined by a 20% or greater, such as 50% or greater, such as 75% orgreater, reduction of the sum of the products of perpendicular diametersof all evaluable tumor nodules or at least 1 month with no tumor sitesshowing enlargement. Similarly, a mixed response can be defined by areduction of the product of perpendicular diameters of all measurablelesions by 20% or greater, such as 50% or greater, with progression inone or more sites.

Of course, the above-described treatment regimes can be altered by thoseof skill in the art, who will be able to take the information disclosedin this specification and optimize treatment regimes.

Example 13 Using Adenosine Receptor Antagonists as an Adjuvant

This example describes a protocol for using one or more adenosinereceptor antagonists as an adjuvant by administering the adenosinereceptor antagonist to a subject in combination with a vaccine. Thisprotocol is intended to serve as an example of such a method, and is notlimiting. Those of skill in the art will be able to modify the protocolto suit the needs of the subject, and to optimize for the particularantagonists and vaccines used.

Adenosine receptor antagonists are administered orally, topically, orparenterally in dosage unit formulations containing standard, well knownnon-toxic physiologically acceptable carriers, adjuvants, and vehiclesas desired. Adenosine receptor antagonists can be administered indosages of about 0.1 mg/kg to about 1 g/kg, depending on the antagonistused. The adenosine receptor antagonists can be delivered to the patientbefore, after or concurrently with the vaccine.

A typical vaccination course can comprise a single dose. Optionally, thecourse can be repeated every twelve weeks or on a more frequent(monthly, weekly, etc.) or less frequent (biannually, annually, everythree years, every ten years, etc.) basis. Of course, these are onlyexemplary times for vaccination, and the skilled practitioner willreadily recognize that many other time-courses are possible. Theadenosine receptor antagonists can be combined with any of a number ofconventional vaccines.

Clinical responses can be defined by an acceptable measure. For example,TNF-α, IFN-γ, IL-4, IL-6, IL-1β and IL-12p40 levels in blood or serumsamples can be determined using commercially available ELISA kitsaccording to manufacturer suggestions. Alternately, antibodies to thevaccine can be measured in blood or serum samples using ELISA kits.

Of course, the above-described treatment regimes can be altered by thoseof skill in the art. In view of the many possible embodiments to whichthe principles of our disclosure may be applied, it should be recognizedthat the illustrated embodiments are only examples of the disclosure andshould not be taken as a limitation on the scope of the disclosure.Rather, the scope of the disclosure is defined by the following claims.We therefore claim as our invention all that comes within the scope andspirit of these claims.

We claim:
 1. A method for treating a tumor in a subject, comprising:administering to the subject a therapeutically effective dose of anadenosine A2a receptor antagonist, or administering to the subject atherapeutically effective dose of an agent at inhibits accumulation ofextracelluar adenosine, or combinations thereof, thereby treating thetumor.
 2. The method of claim 1, further comprising administering to thesubject a therapeutically effective dose of an anti-neoplastic agent. 3.The method of claim 2, wherein the subject is administered theanti-neoplastic agent prior to the adenosine A2a receptor antagonist. 4.The method of claim 2, wherein the anti-neoplastic agent selectivelytargets cells of the tumor.
 5. The method of claim 2, wherein theanti-neoplastic agent comprises an alkylating drug, a folate antagonist,a purine antagonist, a pyrimidine antagonist, a spindle poison, apodophyllotoxin, an antibiotic, a nitrosurea, an inorganic ion, abiologic response modifier, an enzyme, a hormone, an immunotoxin thatselectively binds to a tumor-specific antigen and accumulates in atumor, an agent that has an affinity for tumor cells, or an antibodythat binds a tumor-specific antigen and has antitumor activity in vivo.6. The method of claim 2, wherein the anti-neoplastic agent comprisescyclophosphamide, ifosamide, cisplatin, methotrexate, cytoxan,procarizine, etoposide, BCNU, vincristine, vinblastine,cyclophosphamide, gencitabine, 5-fluorouracie, paclitaxel, ordoxorubicin.
 7. The method of claim 1, further comprising administeringto the subject a therapeutically effective dose of an immunostimulant.8. The method of claim 1, further comprising administering to thesubject a therapeutically effective dose of radiation therapy.
 9. Themethod of claim 1, further comprising administering to the subject atherapeutically effective dose of an immunostimulant and animmunotherapy agent that initiates an immune response directed againstthe tumor.
 10. The method of claim 9, wherein the immunotherapy agentinitiates an immune response directed against the tumor by depletingimmunoregulatory mechanisms that improve anti-tumor CTL activity. 11.The method of claim 10, wherein the immunotherapy agent is a CTLA4ligand or is an agent that removes CD25+ regulatory T cells.
 12. Themethod of claim 1, further comprising administering to the subject atherapeutically effective dose of a monoclonal antibody that binds to atumor-specific antigen and initiates anti-tumor activity in vivo. 13.The method of claim 12, wherein the monoclonal antibody is animmunotoxin.
 14. The method of claim 1, wherein the adenosine A2areceptor antagonist is ZM241385(4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5yl-amino]ethyl)phenol),1,3,7-trimethylxanthine (caffeine), theobromine, SCH 58261, or KW-6002.15. The method of claim 1, wherein the agent that inhibits accumulationof extracelluar adenosine comprises an inhibitor of hypoxia-inducedenzyme.
 16. The method of claim 1, wherein the agent that inhibitsaccumulation of extracelluar adenosine comprises an oxygenation agent, aredox-potential changing agent, adenosine deaminase, adenosine kinase,ADA-PEG, oxygenation of the subject, or combinations thereof.
 17. Themethod of claim 1, wherein the subject previously received chemotherapyor radiotherapy.
 18. The method of claim 1, wherein the subject is ahuman.
 19. The method of claim 1, wherein the subject isimmunosuppressed.
 20. The method of claim 1, wherein the method: reducesa number of tumor cells in the subject; reduces a volume or size of thetumor in the subject; reduces metastasis of the tumor in the subject;reduces a blood supply of a tumor in a subject; or combinations thereof.21. The method of claim 1, wherein the tumor is greater than 2 mm indiameter, and wherein the tumor has local hypoxia areas.
 22. The methodof claim 1, wherein the tumor is a cancer.
 23. The method of claim 18,wherein the cancer is a melanoma, sarcoma, or carcinoma of the colon,breast, cervix, endometrium, ovary, testis, nasopharynx, kidney, lung,skin, or liver.
 24. The method of claim 22, wherein the cancer is acancer of the lung, breast, skin, or liver.